# Patent

## Title
Treating protein folding disorders with small molecule CFTR correctors

## Abstract

Novel CFTR corrector compounds that are effective in rescuing halide efflux in a cell are provided. Also provided are methods for treating protein folding disorders (e.g., cystic fibrosis). The methods include administering a CFTR corrector compound or pharmaceutically acceptable salt or prodrug thereof. Methods of screening for CFTR corrector compounds are also described herein. The methods of screening include contacting a cell that endogenously expresses a CFTR mutation with the compound to be screened and detecting a rescue of halide efflux from the cell.

## Background
CROSS-REFERENCE TO PRIORITY APPLICATIONS
This application claims priority to U.S. Provisional Application No. 61/486,929, filed May 17, 2011, which is incorporated herein by reference in its entirety.


STATEMENT REGARDING FEDERALLY FUNDED RESEARCH
This invention was made with government support under Grant Nos. NIDDK R01 DK060065 and NIDDK Phase I SBIR DK084658 awarded by the National Institutes of Health. The government has certain rights in the invention.


BACKGROUND
Cystic fibrosis is an example of a protein folding disorder. It is a hereditary disease caused by mutations in a gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). The CFTR gene encodes a chloride channel that is expressed in multiple epithelial cell types. A common CFTR mutation, delF508, causes the failure of CFTR to traffic correctly to the plasma membrane because of protein misfolding. The delF508 mutation is estimated to account for 90% of mutant alleles. Because of its high degree of incidence in the cystic fibrosis population, delF508-CFTR is a prime target for cystic fibrosis therapeutics. As such, delF508-CFTR has been extensively studied and is a model for the study of protein folding diseases.
SUMMARY
Compounds and methods for the treatment of protein folding disorders are provided. Cystic fibrosis (CF) is used throughout as an example of such a protein folding disorder. The methods include administering to a subject a CFTR corrector (i.e., a compound effective in rescuing halide efflux in a cell).
A class of CFTR correctors includes compounds of the following structure:




or pharmaceutically acceptable salts and prodrugs thereof. In this class of compounds, R1 and R2 are each independently selected from substituted or unsubstituted alkoxy and substituted or unsubstituted aryloxy; R3 and R4 are each independently selected from hydrogen, halogen, nitro, hydroxyl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryloxy, substituted or unsubstituted amido, substituted or unsubstituted amino, substituted or unsubstituted carbonyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heteroalkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted cycloalkynyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted heterocycloalkenyl, substituted or unsubstituted heterocycloalkynyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; and X1, X2, X3, X4, and X5 are each independently selected from CH and N. In this class of compounds, if X1, X2, X3, X4, and X5 are each CH, R1 and R2 are methoxy, and R4 is hydrogen, then R3 is not hydrogen or chloro.

A class of CFTR correctors includes compounds of the following structure:




or pharmaceutically acceptable salts or prodrugs thereof. In this class of compounds, L is —CH2—, —CH2—CH2—, C═O, or absent; n is 1 or 2; and Z is hydrogen, methyl, or substituted or unsubstituted aryl. Optionally, L is —CH2—. Optionally, L is —CH2—CH2—. Optionally, Z is substituted or unsubstituted aryl.

Also described herein are methods for the treatment of a protein folding disorder in a subject comprising administering to the subject a compound of the following structure:




or pharmaceutically acceptable salts or prodrugs thereof. In this class of compounds, R1, R2, R3, and R4 are each independently selected from hydrogen, halogen, nitro, hydroxyl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryloxy, substituted or unsubstituted amido, substituted or unsubstituted amino, substituted or unsubstituted carbonyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heteroalkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted cycloalkynyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted heterocycloalkenyl, substituted or unsubstituted heterocycloalkynyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; and X1, X2, X3, X4, and X5 are each independently selected from CH and N. Optionally, R1 and R2 are methoxy. Optionally, R3 is chloro. Optionally, X1, X2, X3, X4, and X5 are CH.

In some examples, the compound is:




or pharmaceutically acceptable salts or prodrugs thereof.

A method for the treatment of a protein folding disorder in a subject comprises administering to the subject a compound of the following structure:




and pharmaceutically acceptable salts or prodrugs thereof. In this class of compounds, L is —CH2—, —CH2—CH2—, C═O, or absent; n is 1 or 2; and Z is hydrogen, methyl, or substituted or unsubstituted aryl. Optionally, L is —CH2— or —CH2—CH2—. Optionally, Z is substituted or unsubstituted aryl.

A method for the treatment of a protein folding disorder in a subject comprises administering to the subject a compound of the following structure:




or pharmaceutically acceptable salts or prodrugs thereof. In this class of compounds, R1 and R3 are independently absent or each independently selected from hydrogen or substituted or unsubstituted alkyl; R2 is substituted or unsubstituted amino, substituted or unsubstituted thio, or substituted or unsubstituted heterocycloalkyl; and  is a single bond or double bond, wherein two double bonds are not adjacent. Optionally, R1 and R2 are combined to form a substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.

A method for the treatment of a protein folding disorder in a subject comprises administering to the subject a compound of the following structure:




or pharmaceutically acceptable salts or prodrugs thereof. In this class of compounds, R1 is hydrogen, halogen, alkoxy, or substituted or unsubstituted alkyl; and R2 is hydrogen or substituted or unsubstituted alkyl. Optionally, R1 is ethoxy, chloro, or methyl. Optionally, R2 is methyl.

A method for the treatment of a protein folding disorder in a subject comprises administering to the subject a compound of the following structure:




or pharmaceutically acceptable salts or prodrugs thereof. In this class of compounds, L is NH or piperazine; and R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10 are each independently selected from hydrogen, halogen, nitro, hydroxyl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryloxy, substituted or unsubstituted amido, substituted or unsubstituted amino, substituted or unsubstituted carbonyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heteroalkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted cycloalkynyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted heterocycloalkenyl, substituted or unsubstituted heterocycloalkynyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl. Optionally, R1 is hydrogen or fluoro. Optionally, R2 is hydrogen or chloro. Optionally, R3 is hydrogen, methoxy, or benzyloxy. Optionally, R7 is hydrogen or carboxyl. Optionally, R8 is hydrogen, nitro, or substituted or unsubstituted heteroaryl.

A method for the treatment of a protein folding disorder in a subject comprises administering to the subject a compound of the following structure:




or pharmaceutically acceptable salts or prodrugs thereof. In this class of compounds, R1 is hydrogen or substituted or unsubstituted carbonyl; R2 is hydrogen, carboxyl, or substituted or unsubstituted aryl; R3 is hydrogen, hydroxyl, trifluoromethyl, or substituted or unsubstituted alkyl; and R4 is hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted aryl.

A method for the treatment of a protein folding disorder in a subject comprises administering to the subject a compound of the following structure:




or pharmaceutically acceptable salts or prodrugs thereof. In this class of compounds, L is absent or —CH2—; R1 is substituted or unsubstituted alkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; and R2 is substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl. Optionally, R1 is substituted or unsubstituted phenyl or substituted or unsubstituted quinoline. Optionally, R2 is substituted or unsubstituted phenyl or substituted or unsubstituted furan.

A method for the treatment of a protein folding disorder in a subject comprises administering to the subject a compound of the following structure:




or pharmaceutically acceptable salts or prodrugs thereof. In this class of compounds, R1 is substituted or unsubstituted heterocycloalkenyl or substituted or unsubstituted heteroaryl; R2 and R3 are each independently selected from hydrogen, halogen, trifluoromethyl, or alkoxy; and X is CH or N. Optionally, R1 is substituted or unsubstituted pyrimidinone, substituted or unsubstituted pyrimidine, or substituted or unsubstituted phthalazine. Optionally, R2 is methoxy or trifluoromethyl. Optionally, R3 is chloro or methoxy.

A method for the treatment of a protein folding disorder in a subject comprises administering to the subject a compound of the following structure:




or pharmaceutically acceptable salts or prodrugs thereof. In this class of compounds, R1 is substituted or unsubstituted alkoxy, substituted or unsubstituted aryloxy, substituted or unsubstituted amino, or substituted or unsubstituted piperazine; R2 is hydrogen or substituted or unsubstituted alkyl; and R3 is substituted or unsubstituted alkyl. Optionally, R1 and R2 are combined to form a substituted or unsubstituted heterocycloalkyl or substituted or unsubstituted heterocycloalkenyl.

A method for the treatment of a protein folding disorder in a subject comprises administering to the subject a compound of the following structure:




or pharmaceutically acceptable salts or prodrugs thereof. In this class of compounds, R1, R2, R3, and R4 are each independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; and R5 is hydrogen or substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, or substituted or unsubstituted alkynyl. Optionally, R1 is methyl, ethyl, propyl, allyl, or substituted or unsubstituted benzyl. Optionally, R2 is methyl. Optionally, R3 is methyl, propyl, butyl, isobutyl, allyl, substituted or unsubstituted phenyl, or substituted or unsubstituted benzyl. Optionally, R4 is methyl. Optionally, R5 is hydrogen or methyl.

A method for the treatment of a protein folding disorder in a subject comprises administering to the subject a compound of the following structure:




or pharmaceutically acceptable salts or prodrugs thereof. In this class of compounds, R1 and R2 are each independently selected from hydrogen and substituted or unsubstituted alkyl; R3 is hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted alkenyl; R4 is O or NR5, wherein R5 is substituted or unsubstituted amino or substituted or unsubstituted alkyl; X1 is CH2, O, or NH; and X2 is CH or N. Optionally, X1 is O or CH2. Optionally, R1 and R2 are hydrogen or methyl. Optionally, R3 and R4 are combined to form a substituted or unsubstituted heterocycloalkyl or substituted or unsubstituted heterocycloalkenyl.

A method for the treatment of a protein folding disorder in a subject comprises administering to the subject a compound of the following structure:




or pharmaceutically acceptable salts or prodrugs thereof. In this class of compounds, R1 is hydrogen, halogen, alkoxy, substituted or unsubstituted amino, or substituted or unsubstituted alkyl; and R2, R3, and R4 are each independently substituted or unsubstituted alkyl. Optionally, R1 is chloro, fluoro, or methoxy. Optionally, R2 is methyl. Optionally, R3 is methyl. Optionally, R4 is methyl or ethyl.

Additional or alternative compounds that can be administered to a subject for treating protein folding disorders (e.g., cystic fibrosis) are also provided. The compounds include the following:
















or pharmaceutically acceptable salts and prodrugs thereof. Optionally, the method can further include the step of selecting a subject with cystic fibrosis.

A method of screening for a compound for treating a protein folding disorder (e.g., cystic fibrosis) is also provided. The method includes contacting a cell that endogenously expresses a CFTR mutation with the compound to be screened, and then detecting a rescue of halide efflux from the cell (e.g., using an SPQ assay). The halide efflux rescue indicates that the compound is useful in treating cystic fibrosis. The cell can be, for example, a CF human lung and/or airway epithelial cell. Optionally, the cell is comprised of the CFBE41o-cell line. Optionally, the cell does not overexpress the CFTR mutation. Optionally, the CFTR mutation is delF508-CFTR. Optionally, the halide efflux is chloride efflux. The method of screening can further comprise performing the method with multiple doses of the compound. Optionally, the method of screening can further comprise determining CFTR glycosylation or CFTR immunoprecipitation.



DESCRIPTION OF DRAWINGS
FIG. 1 is a schematic showing a general approach for identifying delF508-CFTR correctors.
FIG. 2 is a schematic of a microtiter plate used for the SPQ high throughput screening assay.
FIG. 3 is a graph demonstrating the dose-response data for daidzein and putative CFCL hit compounds by way of example.
FIG. 4 is a Western blot demonstrating the delF508 CFTR rescue in CF human airway epithelial cells using Compound 43-H11 and related analogs that were validated hit corrector compounds, by way of example. Existing known Corrector drugs 4 and 17, WT-CFTR and DMSO served as the controls. Lane 1 shows the WT-CFTR control. Lanes 2, 3, 4, 5, and 6 represent Compound 43-H11 at concentrations of 10 μM, 1 μM, 100 nM, 10 nM, and 1 nM, respectively. Lane 7 represents the DMSO control. Lanes 8 and 9 represent 10 μM of Corrector 4 and Corrector 7, respectively. Lanes 10 and 11 represent 1 μM of Corrector 4 and Corrector 7, respectively. Lanes 12 and 13 represent 100 nM of Corrector 4 and Corrector 7, respectively. Lane 14 represents the low temperature (27° C.) control.
FIG. 5 is a Western blot demonstrating the delF508 CFTR rescue in CF human airway epithelial cells using Compounds 43-H11 and related analogs, TT NDL 34-H8, and TT NDL 34-A9. Existing known Corrector drugs 4 and 17, WT-CFTR, DMSO, and low temperature (27° C.) served as the controls. Lane 1 shows the WT-CFTR control. Lanes 2, 3, and 4 represent Compound 43-H11 at concentrations of 10 μM, 1 μM, and 100 nM, respectively. Lanes 5, 6, and 7 represent Compound 34-H8 at concentrations of 10 μM, 1 μM, and 100 nM, respectively. Lanes 8, 9, and 10 represent Compound 34A9 at concentrations of 10 μM, 1 μM, and 100 nM, respectively. Lanes 11, 12, and 13 represent Corrector 4 at concentrations of 10 μM, 1 μM, and 100 nM, respectively. Lanes 14, 15, and 16 represent Corrector 17 at concentrations of 10 μM, 1 μM, and 100 nM, respectively. Lane 17 represents the DMSO control. Lane 18 represents the low temperature (27° C.) control.
FIG. 6A is a graph showing the RTE measurements of Compounds 99-A4 and 99-H7 at 2 μM doses. CF delF mouse MLE cell monolayers were used for the tests.
FIG. 6B is a graph showing the RTE measurements of Compounds 99-A4 and 99-H7 at 2 μM doses using CF delF mouse MLE cell monolayers, CLBE41o-, and CF delF MLE cells treated with amiloride and genistein.
FIG. 6C is a graph of RTE measurements demonstrating the dose-response relationships for Compound 99-H7 using CF delF MLE and CFBE41o-cell lines.
FIG. 6D is a Western blot demonstrating the rescue of Band C for Compound 99H7 and Compound 99A4.



DETAILED DESCRIPTION
The compounds and methods described herein are useful in the treatment of protein folding disorders. The compounds and methods described herein can be useful, for example, in the treatment of cystic fibrosis, familial hypercholesterolemia, diabetes mellitus, alpha1 antitrypsin deficiency, Fabry's disease, Gaucher's disease, Pompe's disease, hypothyrosis, and Alzheimer's disease. For example, described herein are compounds and methods useful in the treatment of cystic fibrosis. These compounds are able to correct the misfolding or defective trafficking of delF508-CFTR; thus, the compounds are effective as CFTR correctors (i.e., the compounds are effective in rescuing halide efflux in a cell). Methods for screening for CFTR corrector compounds are also described herein.
A class of CFTR correctors described herein is represented by Formula I:




and pharmaceutically acceptable salts or prodrugs thereof.

In Formula I, R1, R2, R3, and R4 are each independently selected from hydrogen, halogen, nitro, hydroxyl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryloxy, substituted or unsubstituted amido, substituted or unsubstituted amino, substituted or unsubstituted carbonyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heteroalkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted cycloalkynyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted heterocycloalkenyl, substituted or unsubstituted heterocycloalkynyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl. In some examples, R3 is chloro.
In some examples of Formula I, R1 and R2 are each independently selected from substituted or unsubstituted alkoxy and substituted or unsubstituted aryloxy. For example, R1 and R2 are optionally methoxy.
Additionally in Formula I, X1, X2, X3, X4, and X5 are each independently selected from CH and N. In some examples, X1, X2, X3, X4, and X5 are CH.
As used herein, the terms alkyl, alkenyl, and alkynyl include straight- and branched-chain monovalent substituents. Examples include methyl, ethyl, isobutyl, 3-butynyl, and the like. Ranges of these groups useful with the compounds and methods described herein include C1-C20 alkyl, C2-C20 alkenyl, and C2-C20 alkynyl. Additional ranges of these groups useful with the compounds and methods described herein include C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C4 alkyl, C2-C4 alkenyl, and C2-C4 alkynyl.
Heteroalkyl, heteroalkenyl, and heteroalkynyl are defined similarly as alkyl, alkenyl, and alkynyl, but can contain O, S, or N heteroatoms or combinations thereof within the backbone. Ranges of these groups useful with the compounds and methods described herein include C1-C20 heteroalkyl, C2-C20 heteroalkenyl, and C2-C20 heteroalkynyl. Additional ranges of these groups useful with the compounds and methods described herein include C1-C12 heteroalkyl, C2-C12 heteroalkenyl, C2-C12 heteroalkynyl, C1-C6 heteroalkyl, C2-C6 heteroalkenyl, C2-C6 heteroalkynyl, C1-C4 heteroalkyl, C2-C4 heteroalkenyl, and C2-C4 heteroalkynyl.
The terms cycloalkyl, cycloalkenyl, and cycloalkynyl include cyclic alkyl groups having a single cyclic ring or multiple condensed rings. Examples include cyclohexyl, cyclopentylethyl, and adamantanyl. Ranges of these groups useful with the compounds and methods described herein include C3-C20 cycloalkyl, C3-C20 cycloalkenyl, and C3-C20 cycloalkynyl. Additional ranges of these groups useful with the compounds and methods described herein include C5-C12 cycloalkyl, C5-C12 cycloalkenyl, C5-C12 cycloalkynyl, C5-C6 cycloalkyl, C5-C6 cycloalkenyl, and C5-C6 cycloalkynyl.
The terms heterocycloalkyl, heterocycloalkenyl, and heterocycloalkynyl are defined similarly as cycloalkyl, cycloalkenyl, and cycloalkynyl, but can contain O, S, or N heteroatoms or combinations thereof within the cyclic backbone. Ranges of these groups useful with the compounds and methods described herein include C3-C20 heterocycloalkyl, C3-C26 heterocycloalkenyl, and C3-C20 heterocycloalkynyl. Additional ranges of these groups useful with the compounds and methods described herein include C5-C12 heterocycloalkyl, C5-C12 heterocycloalkenyl, C5-C12 heterocycloalkynyl, C5-C6 heterocycloalkyl, C5-C6 heterocycloalkenyl, and C5-C6 heterocycloalkynyl.
Aryl molecules include, for example, cyclic hydrocarbons that incorporate one or more planar sets of, typically, six carbon atoms that are connected by delocalized electrons numbering the same as if they consisted of alternating single and double covalent bonds. An example of an aryl molecule is benzene. Heteroaryl molecules include substitutions along their main cyclic chain of atoms such as O, N, or S. When heteroatoms are introduced, a set of five atoms, e.g., four carbon and a heteroatom, can create an aromatic system. Examples of heteroaryl molecules include furan, pyrrole, thiophene, imadazole, oxazole, pyridine, and pyrazine. Aryl and heteroaryl molecules can also include additional fused rings, for example, benzofuran, indole, benzothiophene, naphthalene, anthracene, and quinoline.
The alkyl, alkenyl, alkynyl, aryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocycloalkyl, heterocycloalkenyl, or heterocycloalkynyl molecules used herein can be substituted or unsubstituted. As used herein, the term substituted includes the addition of an alkyl, alkenyl, alkynyl, aryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocycloalkyl, heterocycloalkenyl, or heterocycloalkynyl group to a position attached to the main chain of the alkyl, alkenyl, alkynyl, aryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocycloalkyl, heterocycloalkenyl, or heterocycloalkynyl, e.g., the replacement of a hydrogen by one of these molecules. Examples of substitution groups include, but are not limited to, hydroxyl, halogen (e.g., F, Br, Cl, or I), and carboxyl groups. For example, substituted alkyl groups can include fluorinated alkyl groups such as 1-fluoromethyl or trifluoromethyl. Conversely, as used herein, the term unsubstituted indicates the alkyl, alkenyl, alkynyl, aryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocycloalkyl, heterocycloalkenyl, or heterocycloalkynyl has a full complement of hydrogens, i.e., commensurate with its saturation level, with no substitutions, e.g., linear decane (—(CH2)9—CH3).
In some examples of Formula I, if X1, X2, X3, X4, and X5 are each CH, R1 and R2 are methoxy, and R4 is hydrogen, then R3 is not hydrogen or chloro.
In some examples, Formula I is represented by Structure I-A:




In Structure I-A, R1 and R2 are substituted or unsubstituted alkoxy or aryloxy groups. In some examples, R1 and R2 are not simultaneously methoxy.
In some examples, Formula I is represented by Structure I-B:




In Structure I-B, R3 is as defined above for Formula I. In some examples, R3 is not hydrogen.
In some examples, Formula I is represented by Structure I-C:




In Structure I-C, R1 and R2 are substituted or unsubstituted alkoxy or aryloxy groups and R3 and R4 are as defined above for Formula I. In some examples, when R3 is chloro and R4 is hydrogen, then R1 and R2 are not simultaneously methoxy.
In some examples, Formula I is represented by Structure I-D:




In Structure I-D, R1, R2, R3, and R4 are as defined above for Formula I.
In some examples, Formula I is represented by Structure I-E:




In Structure I-E, R4 is as defined above for Formula I.
Examples of Formula I include the following compounds:




In some examples of Formula I, the compound is not Compound I-1, Compound I-2, or Compound I-3.
A class of CFTR correctors described herein is represented by Formula II:




and pharmaceutically acceptable salts or prodrugs thereof.

In Formula II, L is —CH2—, —CH2—CH2—, C═O, or absent. Optionally, L is —CH2— or —CH2—CH2—.
Also, in Formula II, n is 1 or 2.
Additionally, in Formula II, Z is hydrogen, methyl, or substituted or unsubstituted aryl. Optionally, Z is substituted or unsubstituted aryl.
In some examples, Formula II is represented by Structure II-A or Structure II-B:




In Structures II-A and II-B, L is as defined above for Formula II.
Examples of Formula II include the following compounds:













A class of CFTR correctors described herein is represented by Formula III:




and pharmaceutically acceptable salts or prodrugs thereof.

In Formula III, R1 and R3 are independently absent or each independently selected from hydrogen or substituted or unsubstituted alkyl.
Also, in Formula III, R2 is substituted or unsubstituted amino, substituted or unsubstituted thio, or substituted or unsubstituted heterocycloalkyl.
Additionally, in Formula III,  is a single bond or double bond, wherein two double bonds are not adjacent.
In some examples of Formula III, R1 and R2 are combined to form a substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl. For example, R1 can be a substituted ethylene group and R2 can be a substituted ethenamine group that combine to form a substituted pyrimidine group.
Examples of Formula III include the following compounds:




A class of CFTR correctors described herein is represented by Formula IV:




and pharmaceutically acceptable salts and prodrugs thereof.

In Formula IV, R1 is hydrogen, halogen, alkoxy, or substituted or unsubstituted alkyl. Optionally, R1 is ethoxy, chloro, or methyl.
Also, in Formula IV, R2 is hydrogen or substituted or unsubstituted alkyl. Optionally, R2 is methyl.
Examples of Formula IV include the following compounds:




A class of CFTR correctors described herein is represented by Formula V:




and pharmaceutically acceptable salts or prodrugs thereof.

In Formula V, L is NH or piperazine.
Also, in Formula V, R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10 are each independently selected from hydrogen, halogen, nitro, hydroxyl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryloxy, substituted or unsubstituted amido, substituted or unsubstituted amino, substituted or unsubstituted carbonyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heteroalkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted cycloalkynyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted heterocycloalkenyl, substituted or unsubstituted heterocycloalkynyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl. In some examples, R1 is hydrogen or fluoro. In some examples, R2 is hydrogen or chloro. In some examples, R3 is hydrogen, methoxy, or benzyloxy. In some examples, R7 is hydrogen or carboxyl. In some examples, R8 is hydrogen, nitro, or substituted or unsubstituted heteroaryl.
Examples of Formula V include the following compounds:




A class of CFTR correctors described herein is represented by Formula VI:




and pharmaceutically acceptable salts or prodrugs thereof.

In Formula VI, R1 is hydrogen or substituted or unsubstituted carbonyl.
Also, in Formula VI, R2 is hydrogen, carboxyl, or substituted or unsubstituted aryl.
Additionally, in Formula VI, R3 is hydrogen, hydroxyl, trifluoromethyl, or substituted or unsubstituted alkyl.
Further, in Formula VI, R4 is hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted aryl.
Examples of Formula VI include the following compounds:




A class of CFTR correctors described herein is represented by Formula VII:




and pharmaceutically acceptable salts or prodrugs thereof.

In Formula VII, L is absent or —CH2—.
Also, in Formula VII, R1 is substituted or unsubstituted alkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl. Optionally, R1 is substituted or unsubstituted phenyl or substituted or unsubstituted quinoline.
Additionally, in Formula VII, R2 is substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl. Optionally, R2 is substituted or unsubstituted phenyl or substituted or unsubstituted furan.
Examples of Formula VII include the following compounds:







A class of CFTR correctors described herein is represented by Formula VIII:




and pharmaceutically acceptable salts or prodrugs thereof.

In Formula VIII, R1 is substituted or unsubstituted heterocycloalkenyl or substituted or unsubstituted heteroaryl. Optionally, R1 is substituted or unsubstituted pyrimidinone, substituted or unsubstituted pyrimidine, or substituted or unsubstituted phthalazine.
Also, in Formula VIII, R2 and R3 are each independently selected from hydrogen, halogen, trifluoromethyl, or alkoxy. Optionally, R2 is methoxy or trifluoromethyl. Optionally, R3 is chloro or methoxy.
Additionally, in Formula VIII, X is CH or N.
Examples of Formula VIII include the following compounds:







A class of CFTR correctors described herein is represented by Formula IX:




and pharmaceutically acceptable salts or prodrugs thereof.

In Formula IX, R1 is substituted or unsubstituted alkoxy, substituted or unsubstituted aryloxy, substituted or unsubstituted amino, or substituted or unsubstituted piperazine.
Also, in Formula IX, R2 is hydrogen or substituted or unsubstituted alkyl.
Additionally, in Formula IX, R3 is substituted or unsubstituted alkyl.
In some examples, R1 and R2 are combined to form a substituted or unsubstituted heterocycloalkyl or substituted or unsubstituted heterocycloalkenyl.
Examples of Formula IX include the following compounds:




A class of CFTR correctors described herein is represented by Formula X:




and pharmaceutically acceptable salt or prodrug thereof.

In Formula X, R1, R2, R3, and R4 are each independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl. Optionally, R1 is methyl, ethyl, propyl, allyl, or substituted or unsubstituted benzyl. Optionally, R2 is methyl. Optionally, R3 is methyl, propyl, butyl, isobutyl, allyl, substituted or unsubstituted phenyl, or substituted or unsubstituted benzyl. Optionally, R4 is methyl.
Also, in Formula X, R5 is hydrogen or substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, or substituted or unsubstituted alkynyl. Optionally, R5 is hydrogen or methyl.
Examples of Formula X include the following compounds:
















A class of CFTR correctors described herein is represented by Formula XI:




and pharmaceutically acceptable salts or prodrugs thereof.

In Formula XI, R1 and R2 are each independently selected from hydrogen and substituted or unsubstituted alkyl. Optionally, R1 and R2 are hydrogen or methyl.
Also, in Formula XI, R3 is hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted alkenyl.
Additionally, in Formula XI, R4 is O or NR5, wherein R5 is substituted or unsubstituted amino or substituted or unsubstituted alkyl.
Also, in Formula XI, X1 is CH2, O, or NH.
Further, in Formula XI, X2 is CH or N.
In some examples of Formula XI, R3 and R4 are combined to form a substituted or unsubstituted heterocycloalkyl or substituted or unsubstituted heterocycloalkenyl.
Examples of Formula XI include the following compounds:




A class of CFTR correctors described herein is represented by Formula XII:




and pharmaceutically acceptable salts or prodrugs thereof.

In Formula XII, R1 is hydrogen, halogen, alkoxy, substituted or unsubstituted amino, or substituted or unsubstituted alkyl. Optionally, R1 is chloro, fluoro, or methoxy.
Also, in Formula XII, R2, R3, and R4 are each independently substituted or unsubstituted alkyl. Optionally, R2 is methyl. Optionally, R3 is methyl. Optionally, R4 is methyl or ethyl.
Examples of Formula XII include the following compounds:




Additional compounds useful with the methods described herein include the following Compounds 1-28:
















The methods described herein include a method of treating protein folding disorders (e.g., cystic fibrosis) in a subject. These methods include the step of administering to the subject a compound of the structures described herein. Additional steps can be included in the method described herein. For example, the methods can further include the steps of selecting a subject with a protein folding disorder, such as cystic fibrosis, and administering to the subject one or more of the CFTR correctors described herein.
The compounds described herein can be prepared in a variety of ways. The compounds can be synthesized using various synthetic methods. At least some of these methods are known in the art of synthetic organic chemistry. The compounds described herein can be prepared from readily available starting materials. Optimum reaction conditions can vary with the particular reactants or solvent used, but such conditions can be determined by one skilled in the art by routine optimization procedures.
Variations on Formulas I-XII and Compounds 1-28 include the addition, subtraction, or movement of the various constituents as described for each compound. Similarly, when one or more chiral centers are present in a molecule, the chirality of the molecule can be changed. Additionally, compound synthesis can involve the protection and deprotection of various chemical groups. The use of protection and deprotection, and the selection of appropriate protecting groups can be determined by one skilled in the art. The chemistry of protecting groups can be found, for example, in Greene, et al., Protective Groups in Organic Synthesis, 2d. Ed., Wiley & Sons, 1991, which is incorporated herein by reference in its entirety.
Reactions to produce the compounds described herein can be carried out in solvents, which can be selected by one of skill in the art of organic synthesis. Solvents can be substantially nonreactive with the starting materials (reactants), the intermediates, or products under the conditions at which the reactions are carried out, i.e., temperature and pressure. Reactions can be carried out in one solvent or a mixture of more than one solvent. Product or intermediate formation can be monitored according to any suitable method known in the art. For example, product formation can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g., 1H or 13C) infrared spectroscopy, spectrophotometry (e.g., UV-visible), or mass spectrometry, or by chromatography such as high performance liquid chromatograpy (HPLC) or thin layer chromatography.
One or more of the compounds described herein or pharmaceutically acceptable salts or prodrugs thereof can be provided in a pharmaceutical composition comprising a pharmaceutical carrier. Furthermore, the one or more compounds described herein can be combined with other agents, including treatments for lung, digestive, hepatic, and biliary tract related diseases and disorders. For example, in the case of cystic fibrosis, the compounds described herein can be combined with mucus thinning drugs (e.g., dornase alfa, N-Acetyl cysteine, and hypertonic saline), bronchodilators (e.g., metaproterenol sulfate, pirbuterol acetate, salmeterol, albuterol, and terbutaline sulfate), P2Y2-receptor agonists (e.g., denufosol), and agents that target nonsense mutations (e.g., PTC124). Further examples of additional agents that can be combined with the compounds described herein include antibiotics (e.g., aminoglycosides, antipseudomonal penicillins, and cephalosporins), antimicrobial drugs (e.g., rifabutin), ethambutol, clarithromycin, clofazimine, aztreonam, steroidal and nonsteroidal anti-inflammatory drugs (e.g., ibuprofen and prednisone), pentoxifylline, dornase alfa, or ursodeoxycholic acid.
The one or more compounds described herein can be provided as pharmaceutical compositions administered in combination with one or more other therapeutic or prophylactic agents. As used throughout, a therapeutic agent is a compound or composition effective in ameliorating a pathological condition. Illustrative examples of therapeutic agents include, but are not limited to, chemotherapeutic agents, anti-viral agents, anti-opportunistic agents, antibiotics, and immunostimulatory agents. Optionally, more than one therapeutic agent is administered in combination with the provided compositions.
The one or more compounds described herein, with or without additional agents, can be provided in the form of an inhaler or nebulizer for inhalation therapy. As used herein, inhalation therapy refers to the delivery of a therapeutic agent, such as the compounds described herein, in an aerosol form to the respiratory tract (i.e., pulmonary delivery). Additional inhalants useful for delivery of the compounds described herein include intra-oral sprays, mists, metered dose inhalers, and dry powder generators (See Gonda, J. Pharm. Sci. 89:940-945, 2000, which is incorporated herein by reference in its entirety, at least, for inhalation delivery methods taught therein).
Depending on the intended mode of administration, the pharmaceutical composition can be in the form of solid, semi-solid, or liquid dosage forms, such as, for example, tablets, suppositories, pills, capsules, powders, liquids, aerosols, or suspensions, preferably in unit dosage form suitable for single administration of a precise dosage. The compositions will include a therapeutically effective amount of the compound described herein or derivatives thereof in combination with a pharmaceutically acceptable carrier and, in addition, can include other medicinal agents, pharmaceutical agents, carriers, or diluents. By pharmaceutically acceptable is meant a material that is not biologically or otherwise undesirable, which can be administered to an individual along with the selected compound without causing unacceptable biological effects or interacting in a deleterious manner with the other components of the pharmaceutical composition in which it is contained.
As used herein, the term carrier encompasses any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations. The choice of a carrier for use in a composition will depend upon the intended route of administration for the composition. The preparation of pharmaceutically acceptable carriers and formulations containing these materials is described in, e.g., Remington's Pharmaceutical Sciences, 21st Edition, ed. University of the Sciences in Philadelphia, Lippincott, Williams & Wilkins, Philadelphia Pa., 2005. Examples of physiologically acceptable carriers include buffers such as phosphate buffers, citrate buffer, and buffers with other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN® (ICI, Inc.; Bridgewater, N.J.), polyethylene glycol (PEG), and PLURONICSTM (BASF; Florham Park, N.J.).
Compositions containing the compound described herein or pharmaceutically acceptable salts or prodrugs thereof suitable for parenteral injection can comprise physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propyleneglycol, polyethyleneglycol, glycerol, and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.
These compositions can also contain adjuvants such as preserving, wetting, emulsifying, and dispensing agents. Prevention of the action of microorganisms can be promoted by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. Isotonic agents, for example, sugars, sodium chloride, and the like can also be included. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.
Solid dosage forms for oral administration of the compounds described herein or pharmaceutically acceptable salts or prodrugs thereof include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the compounds described herein or derivatives thereof is admixed with at least one inert customary excipient (or carrier) such as sodium citrate or dicalcium phosphate or (a) fillers or extenders, as for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid, (b) binders, as for example, carboxymethylcellulose, alignates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, (c) humectants, as for example, glycerol, (d) disintegrating agents, as for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate, (e) solution retarders, as for example, paraffin, (f) absorption accelerators, as for example, quaternary ammonium compounds, (g) wetting agents, as for example, cetyl alcohol, and glycerol monostearate, (h) adsorbents, as for example, kaolin and bentonite, and (i) lubricants, as for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets, and pills, the dosage forms can also comprise buffering agents.
Solid compositions of a similar type can also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethyleneglycols, and the like.
Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings and others known in the art. They can contain opacifying agents and can also be of such composition that they release the active compound or compounds in a certain part of the intestinal tract in a delayed manner. Examples of embedding compositions that can be used are polymeric substances and waxes. The active compounds can also be in micro-encapsulated form, if appropriate, with one or more of the above-mentioned excipients.
Liquid dosage forms for oral administration of the compounds described herein or pharmaceutically acceptable salts or prodrugs thereof include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compounds, the liquid dosage forms can contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents, and emulsifiers, as for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butyleneglycol, dimethylformamide, oils, in particular, cottonseed oil, groundnut oil, corn germ oil, olive oil, castor oil, sesame oil, glycerol, tetrahydrofurfuryl alcohol, polyethyleneglycols, and fatty acid esters of sorbitan, or mixtures of these substances, and the like.
Besides such inert diluents, the composition can also include additional agents, such as wetting, emulsifying, suspending, sweetening, flavoring, or perfuming agents.
Suspensions, in addition to the active compounds, can contain additional agents, as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances, and the like.
Compositions of the compounds described herein or pharmaceutically acceptable salts or prodrugs thereof for rectal administrations are optionally suppositories, which can be prepared by mixing the compounds with suitable non-irritating excipients or carriers such as cocoa butter, polyethyleneglycol or a suppository wax, which are solid at ordinary temperatures but liquid at body temperature and therefore, melt in the rectum or vaginal cavity and release the active component.
Dosage forms for topical administration of the compounds described herein or pharmaceutically acceptable salts or prodrugs thereof include ointments, powders, sprays, aerosols, and inhalants (e.g., intra-oral sprays, mists, metered dose inhalers, nebulizers, and dry powder generators). The compounds described herein or pharmaceutically salts or prodrugs thereof are admixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants as can be required. Ophthalmic formulations, ointments, powders, and solutions are also contemplated as being within the scope of the compositions.
The term pharmaceutically acceptable salts as used herein refers to those salts of the compound described herein or derivatives thereof that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of subjects without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use, as well as the zwitterionic forms, where possible, of the compounds described herein. The term salts refers to the relatively non-toxic, inorganic and organic acid addition salts of the compounds described herein. These salts can be prepared in situ during the isolation and purification of the compounds or by separately reacting the purified compound in its free base form with a suitable organic or inorganic acid and isolating the salt thus formed. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate mesylate, glucoheptonate, lactobionate, methane sulphonate, and laurylsulphonate salts, and the like. These can include cations based on the alkali and alkaline earth metals, such as sodium, lithium, potassium, calcium, magnesium, and the like, as well as non-toxic ammonium, quaternary ammonium, and amine cations including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. (See Stahl and Wermuth, Pharmaceutical Salts: Properties, Selection, and Use, Wiley-VCH, 2008, which is incorporated herein by reference in its entirety, at least, for compositions taught therein).
Administration of compounds described herein or pharmaceutically acceptable salts or prodrugs thereof can be carried out using therapeutically effective amounts of the compounds described herein or pharmaceutically acceptable salts or prodrugs thereof for periods of time effective to treat neurological disorders. The effective amount of the compounds described herein or pharmaceutically acceptable salts or prodrugs thereof can be determined by one of ordinary skill in the art and includes exemplary dosage amounts for a mammal of from about 0.5 to about 200 mg/kg of body weight of active compound per day, which can be administered in a single dose or in the form of individual divided doses, such as from 1 to 4 times per day. Alternatively, the dosage amount can be from about 0.5 to about 150 mg/kg of body weight of active compound per day, about 0.5 to 100 mg/kg of body weight of active compound per day, about 0.5 to about 75 mg/kg of body weight of active compound per day, about 0.5 to about 50 mg/kg of body weight of active compound per day, about 0.5 to about 25 mg/kg of body weight of active compound per day, about 1 to about 20 mg/kg of body weight of active compound per day, about 1 to about 10 mg/kg of body weight of active compound per day, about 20 mg/kg of body weight of active compound per day, about 10 mg/kg of body weight of active compound per day, or about 5 mg/kg of body weight of active compound per day. Those of skill in the art will understand that the specific dose level and frequency of dosage for any particular subject can be varied and will depend upon a variety of factors, including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the species, age, body weight, general health, sex and diet of the subject, the mode and time of administration, rate of excretion, drug combination, and severity of the particular condition.
In the methods described herein, the subjects treated can be further treated with one or more additional agents. The one or more additional agents and the compounds described herein or pharmaceutically acceptable salts or prodrugs thereof can be administered together in a single composition (e.g., as an admixture) or in separate compositions in any order, including simultaneous administration, as well as temporally spaced order of up to several days apart. The methods can also include more than a single administration of the one or more additional agents and/or the compounds described herein or pharmaceutically acceptable salts or prodrugs thereof. The administration of the one or more additional agents and the compounds described herein or pharmaceutically acceptable salts or prodrugs thereof can be by the same or different routes and concurrently or sequentially. By treatment, treating, or treat is meant a reduction in one or more signs or symptoms or an improvement in the clinical state of the subject being treated for a disease or disorder (e.g., cystic fibrosis). For example, reduced numbers of infections or hospitalizations, reduction in respiratory or gastrointestinal symptoms, improved nutritional status, or improved pulmonary function indicate effective treatment.
As described above, the compounds described herein are useful in the treatment of protein folding disorders. Examples of protein folding disorders include cystic fibrosis; neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Creutzfeld-Jakob disease, Kuru, GSS disease, Huntington's disease, Polyglutamine diseases, Prion disease, Bovine Spongiform Encephalopathy (BSE), Amyotrophic Lateral Sclerosis, Alexander's disease, Primary Systemic Amyloidosis, Secondary Systemic Amyloidosis, Senile Systemic Amyloidosis, and Amyloidosis in senescence; ocular diseases such as Cataract, Retinitis Pigmentosa, and Macular Degeneration; and other diseases such as Islet amyloid, Medullar Carcinoma of the Thyroid, Hereditary Renal Amyloidosis, Hemodialysis-related amyloidosis, Desmin-related Cardiomyopathy, Charcot-Marie Tooth disease, diabetes insipidis, diabetes insipidis, alpha1 antitrypsin deficiency, Fabry's disease, Gaucher's disease, Pompe's disease, and Charcot-Marie Tooth disease.
Additionally, a method of screening for a compound for treating cystic fibrosis (i.e., a CFTR corrector) is provided. Compounds that are suitable for treating cystic fibrosis have the ability to rescue delF508-CFTR. In addition, the compounds are effective on endogenously expressed delF508-CFTR. Further, the compounds are stable and the effects persist in serum-containing medium. The method for screening for CFTR correctors involves the steps of contacting a cell with the compound to be screened and detecting rescue of halide efflux from the cell. The halide efflux rescue indicates that the compound is useful in treating cystic fibrosis (i.e., the compound is a CFTR corrector). The method can be performed in vitro or in vivo. The method provides an effective and reliable means of screening for CFTR correctors.
The cell models used in other methods of identifying CFTR correctors have employed low temperature, chemical chaperones such as glycerol, 4-phenylbutyrate, DMSO, and overexpression of CFTR in a transduced Fisher rat thyroid cell line as the model, whereas the present methods do not require, and optionally exclude, over-expression of CFTR, low temperature, and chemical chaperones, variables that can distort the results.
The cells useful with the methods described herein endogenously express a CFTR mutation (e.g., the delF508-CFTR mutation). In some examples, the cell can be a human airway epithelial cell (e.g., CFBE41o-cells). CFBE41o-cells are human airway epithelial cells on a delF508-CFTR homozygous background. An example of cells useful with the methods described herein includes cells that do not overexpress the CFTR mutation.
The step of detecting a rescue of halide efflux from the cell can be monitored using the halide quenched dye 6-methoxy-N-(3-sulfopropyl)-quinolinium (SPQ, Molecular Probes Inc., Eugene, Oreg.), as described below in Example 1. In some examples, the halide efflux monitored can be chloride efflux.
The method of screening for a compound for treating cystic fibrosis as described herein can be performed with multiple doses of the compound as shown in Example 2. The method of screening for a compound for treating cystic fibrosis as described herein can further include determining CFTR immunoprecipitation, as described in Example 3. In addition, the method of screening for a compound for treating cystic fibrosis as described herein can also include determining CFTR glycosylation, as described in Example 4. CFTR glycosylation is determined by monitoring the two N-linked glycosylation sites on the fourth extracellular loop of CFTR. The immature, ER form shows as Band B on an SDS-PAGE gel, while a fully processed, mature form appears as Band C on an SDS-PAGE gel. Agents that correct ΔF508 CFTR promote Band C formation. Thus, CFTR glycosylation can be used to monitor correction.
The CFTR glycosylation assay can be used to determine whether the protein is in a post-Golgi compartment. However, this assay does not determine whether the protein is on the cell surface. Thus, the CFTR glycosylation assay can be used in conjunction with surface biotinylation, which does display whether the protein is on the cell surface. The CFTR glycosylation assay and surface biotinylation assays can used as complementary approaches for illustrating the effectiveness of ΔF508 CFTR correction.
In general, compounds useful for treating cystic fibrosis can be identified from large libraries of natural products or synthetic (or semi-synthetic) extracts or chemical libraries according to methods known in the art. The precise source of test extracts or compounds is not critical to the screening procedure(s). Accordingly, virtually any number of chemical extracts or compounds can be screened using the methods described herein. Examples of such extracts or compounds include, but are not limited to, plant-, fungal-, prokaryotic- or animal-based extracts, fermentation broths, and synthetic compounds, as well as modifications of existing compounds. Numerous methods are also available for generating random or directed synthesis (e.g., semi-synthesis or total synthesis) of any number of chemical compounds, including, but not limited to, saccharide-, lipid-, peptide-, polypeptide- and nucleic acid-based compounds. Synthetic compound libraries and libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are commercially available. In addition, natural and synthetically produced libraries are generated, if desired, according to methods known in the art, e.g., by standard extraction and fractionation methods. Furthermore, if desired, any library or compound is readily modified using standard chemical, physical, or biochemical methods.
The examples below are intended to further illustrate certain aspects of the methods and compounds described herein, and are not intended to limit the scope of the claims.
EXAMPLES
Example 1
SPQ High Throughput Screening Assay
A schematic showing a general approach for identifying delF508-CFTR correctors is shown in FIG. 1. In FIG. 1, a non-CF ciliated airway epithelial cell is shown at left and a CF airway cell is shown at right. In CF, chloride secretion is often lost and sodium absorption becomes hyperactive. As a result, water is absorbed too vigorously in CF due to osmotic forces. Dehydration of the airway surface liquid causes a “domino effect” of problems with ciliary beat, mucociliary clearance, accumulation of dehydrated mucus and infection with opportunistic bacteria. The green and orange color depicts non-mucoid and mucoid Pseudomonas aeruginosa bacteria, respectively, that have taken hold and are accumulating within the thickened mucus in progressing CF pulmonary disease. The approach for discovering delF508-CFTR correctors is shown in FIG. 1 in the right panel.
To perform the SPQ high throughput screening assay, CFBE41o-cells are seeded into 96-well microtiter plates and are loaded with the fluorescent halide-sensitive dye, SPQ, in serum-containing culture medium. Certain wells are loaded with daidzein or hesperetin, which are known flavanoid positive control corrector molecules. For example, as shown in FIG. 2, daidzein is loaded into wells corresponding to A4, A5, and A6. The test compounds are loaded into wells and are tested in triplicate wells (e.g., wells A7, A8, and A9 in FIG. 2) at a 10 nM dose and incubated over 48 hours at room temperature. During the 48 hour period, SPQ is absorbed. Plates are washed in a sodium chloride (NaCl) based Ringer and read once over two minutes to set the baseline SPQ fluorescence activity. Then, NaCl is replaced by sodium nitrate (NaNO3) based Ringer. The plates are read twice over four minutes. The primary high throughput screen (HTS) data are analyzed to detect any function of rescued delF508-CFTR under basal conditions. NaNO3 based Ringer containing genistein (25 μM) is then added and the plates are read five times over ten minutes to determine the delF508-CFTR activity that can be rescued and active on the cell surface. Genistein is then removed, and NaNO3 based Ringer is maintained. The plate is read up to two times to complete the SPQ HTS assay.
Example 2
Dose Response Assessment
The method described in Example 1 is repeated with doses of the test compound of 0.1 μM, 1 μM, 10 μM, and 50 μM in Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) for 48 hours at 37° C. Unaltered CFBE41o-cells are used as the model. A Western blot analysis of the lysates (10-50 ng total protein) will be performed using the MM13-4 antibody for human CFTR to monitor changes in the CFTR protein. The most effective concentrations and time courses (12-96 h) are determined. By way of example, a graph showing the dose response results for exemplary compounds is provided in FIG. 3. Daidzein was used as the control.
Example 3
CFTR Immunoprecipitation
CFTR is immunoprecipitated under mild detergent conditions (1% digitonin, 2.5 mM HEPES, 10.0 mM CaCl2, pH 7.6). The isolated protein complexes are run on SDS-PAGE gels and analyzed by mass spectroscopy. The CFBE41o-control cells are lysed in 2% digitonin (2.5 mM HEPES, 10.0 mM CaCl2, pH 7.6). All lysis buffers are supplemented with a protease inhibitor cocktail (Complete Mini, Roche, Nutley, N.J.). CFTR is immunoprecipitated using Protein A-immobilized agarose beads and antibodies to the C-terminus of CFTR or to the second nucleotide-binding domain. The antibodies are covalently coupled to agarose beads before use (PROFOUND Mammalian Co-IP Kit, Pierce, Rockford, Ill.). The immunoprecipitated CFTR complexes are run on gels, and the interacting proteins are analyzed by mass spectroscopy.
Example 4
CFTR Surface Biotinylation
The cell surfaces are biotinylated and the cells are homogenized. The membrane fragments are isolated and lysed in co-immunoprecipitation buffer. The biotinylated protein complexes are captured with avidin, followed by coupling of the anti-CFTR antibody to agarose beads. The intact protein complexes are separated following CFTR immunoprecipitation during electrophoresis. Cells with no biotin and cells biotinylated but without CFTR expression are used as the controls.
Example 5
CFTR Glycosylation
Cells are lysed in RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% Deoxycholic Acid, 0.1% SDS) plus a cocktail of protease inhibitors (Roche; Basel, Switzerland). Protein concentrations in the cell lysates are measured by BCA Protein Assay using BSA as standard (Pierce; Rockford, Ill.). Proteins (25 μg) are resolved on a 8% SDS-PAGE gel and transferred to PDVF membranes. Total CFTR in the lysate is detected by immunoblotting using a specific CFTR antibody (MM13-4 from Upstate, 1:500 dilution).
Example 6
Biochemical CFTR Rescue with delF508-CFTR Corrector Ligands
The compounds were then subjected to a biochemical assay to define which hit compounds rescued the band B core glycosylated endoplasmic reticulum (ER) form of delF508-CFTR within the cell interior into the maturely glycosylated band C form within the secretory pathway for proteins and within the plasma membrane (FIG. 4). Effective compounds stabilized the band B form of CFTR and caused more of this form to accumulate at the level of the ER. The most effective compounds caused the band C form to appear.
By way of example, compound 43H11, a validated CFTR Corrector compound, was added to CFBE41o-ΔF cells in OptiMEM-1 containing 2% serum in amounts of 10 μM, 1 μM, 100 nM, 10 nM, and 1 nM (see FIG. 4). DMSO, Corrector 4, Corrector 17, and WT-CFTR served as controls. A low temperature (27° C.) correction control was also used. Correctors 4 and 17 are known CFTR correctors available to the CF research community. The media and compound were replenished at 24 hours during the 48 hour treatment. The 1 μM dose of Compound CB 43-H11 was superior to the 10 μM doses of Corrector 4 and Corrector 17, two agents previously found to correct delF508. The 10 μM dose of CB 43-H11 provided similar levels of Band C to that of a low temperature correction control and a WT-CFTR expressing cell control. The delF508-CFTR mutation can be rescued from the ER with low temperature incubation for 48 hours.
Compounds 43-H11, TT NDL 34-H8, and RR NDL 34-A9 were also tested in the biochemical rescue assay. Each compound was tested at 10 μM, 1 μM, and 100 nM. DMSO, Corrector 4, Corrector 17, WT-CFTR, and low temperature (27° C.) served as the controls. (see FIG. 5). The experiments were performed in 10% serum containing medium using the method as described above. The data demonstrate that the corrector compounds described herein are effective independent of serum protein.
Example 7
Electrical Measurements of Transepithelial Resistance
Electrical assay were also performed to determine the functional rescue of delF508-CFTR to the apical cell membrane in a polarized epithelium using the compounds described herein. These experiments were performed using a mouse mixed lung epithelial cell line engineered from the delF508-CFTR mouse (FIG. 6). Compounds 99-A4 and 99-H7 were tested at 2 μM doses. DMSO, WT-CFBE, and delF-CFBE at 37° C. and 27° C. served as the controls.
Only high-resistance cell monolayers (>3,000 Ohms per cm2) were used in the experiments. The RTE measurements shown in FIG. 6A demonstrate that the cell monolayers continue to “tighten” or develop in the presence of delF508-CFTR corrector small molecules. In the presence of amiloride (100 μM), used to block ENaC sodium channels (a step that increased RTE), genistein (25 μM) was added to selectively open any delF508-CFTR chloride channels in the apical cell membrane (FIG. 6B). A significant decrease in RTE suggests that delF508-CFTR chloride channels are opening in the membrane. RTE measurements under voltage clamping show that small molecule 99-A7 is particularly potent. Dose-response relationships showing that 99-A7 rescues functional delF508-CFTR at nanomolar to low micromolar doses after a 48 hour treatment are provided in FIG. 6C.
In this electrical assay on polarized CF human airway epithelial cell monolayers, correction of the Cl— transport defect was shown using a drop in transepithelial resistance as an indicator of rescued delF508-CFTR Cl— channels opening at the cell surface. Compound CB 99-H7 worked well while CB 99-A4 was less effective. Importantly, the transepithelial resistance continued to improve and the monolayers became more mature in the presence of these compounds. The rescue of band C blot is also shown for these compounds (FIG. 6D).
The compounds and methods of the appended claims are not limited in scope by the specific compounds and methods described herein, which are intended as illustrations of a few aspects of the claims and any compounds and methods that are functionally equivalent are within the scope of this disclosure. Various modifications of the compounds and methods in addition to those shown and described herein are intended to fall within the scope of the appended claims. Further, while only certain representative compounds, methods, and aspects of these compounds and methods are specifically described, other compounds and methods are intended to fall within the scope of the appended claims. Thus, a combination of steps, elements, components, or constituents can be explicitly mentioned herein; however, all other combinations of steps, elements, components, and constituents are included, even though not explicitly stated.

## Claims

1. A method for the treatment of a protein folding disorder in a subject, comprising:
administering to the subject a CFTR corrector compound, wherein the CFTR corrector compound has the following structure:




or a pharmaceutically acceptable salt thereof, wherein:
R1 is hydrogen, halogen, alkoxy, substituted or unsubstituted amino, or substituted or unsubstituted alkyl; and
R2, R3, and R4 are each independently substituted or unsubstituted alkyl.

2. The method of claim 1, wherein the protein folding disorder is cystic fibrosis.

3. The method of claim 1, wherein R1 is chloro, fluoro, or methoxy.

4. The method of claim 1, wherein R2 is methyl.

5. The method of claim 1, wherein R3 is methyl.

6. The method of claim 1, wherein R4 is methyl or ethyl.

7. The method of claim 1, wherein the compound is

8. The method of claim 1, wherein the compound is

9. The method of claim 1, wherein the compound is

10. The method of claim 1, wherein the compound is

11. A method for the treatment of a protein folding disorder in a subject, comprising:
administering to the subject a CFTR corrector compound, wherein the CFTR corrector compound has the following structure:





or a pharmaceutically acceptable salt thereof, wherein:
R1 and R3 are independently absent or each independently selected from hydrogen or substituted or unsubstituted alkyl;
R2 is substituted or unsubstituted amino, substituted or unsubstituted thio, or substituted or unsubstituted heterocycloalkyl; and
 is a single bond or double bond, wherein two double bonds are not adjacent,
wherein optionally R1 and R2 are combined to form a substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.

12. A method for the treatment of a protein folding disorder in a subject, comprising:
administering to the subject a CFTR corrector compound, wherein the CFTR corrector compound has the following structure:





or a pharmaceutically acceptable salt thereof, wherein:
R1 is hydrogen or substituted or unsubstituted carbonyl;
R2 is hydrogen, carboxyl, or substituted or unsubstituted aryl;
R3 is hydrogen, hydroxyl, trifluoromethyl, or substituted or unsubstituted alkyl; and
R4 is hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted aryl.

13. A method for the treatment of a protein folding disorder in a subject, comprising:
administering to the subject a CFTR corrector compound, wherein the CFTR corrector compound has the following structure:





or a pharmaceutically acceptable salt thereof.

14. A method for the treatment of a protein folding disorder in a subject, comprising:
administering to the subject a CFTR corrector compound, wherein the CFTR corrector compound has the following structure:





or a pharmaceutically acceptable salt thereof.