Thiazoles and oxazoles useful as modulators of atp-binding cassette transporters

ES 2 328 824 T3

DESCRIPTION

Thiazoles and oxazoles useful as modulators of ATP-binding cassette-type transporters.

Technical field of the invention

The present invention relates to ATP-binding cassette-type transporter modulators or fragments thereof, including the cystic fibrosis transmembrane conductance regulator ("CFTR"), compositions thereof, and related procedures. . The present invention also relates to methods of treating diseases mediated by the ABC transporter using said modulators.

Background of the invention

ABC transporters are a group of membrane transporter proteins that play a fundamental role in the transport and protection of cells against a wide variety of pharmacological agents, potentially toxic drugs, and xenobiotics. ABC transporters are homologous membrane proteins that bind to and use cellular adenosine triphosphate (ATP) for their specific activities. Some of these transporters were discovered as multidrug resistance proteins (such as the MDR1-P glycoprotein, or the multidrug resistance protein, MRP1), which defend malignant cancer cells against chemotherapeutic agents. Almost to date, 48 human ABC transporters have been identified, and these have been arranged into 7 families based on their sequence identity and function.

ABC transporters play several important physiological roles within the body, and they also provide a defense against harmful compounds in the environment. Furthermore, they represent important potential target drugs both in their own right, as well as, because in many cases therapeutic drugs are also transported out of the target cell by these molecules.

One of the members of the ABC transporter family, specifically, CFTR, is believed to be the chloride channel responsible for cAMP-mediated chloride secretion in epithelial cells, and to play a key role in chloride secretion and in maintaining normal electrolyte transport through the body. CFTR is a protein of approximately 1480 amino acids made up of two repeat elements, each of which comprises six transmembrane segments and a nucleotide-binding domain. The two repeat elements are separated by a large polar regulatory (R) domain that contains multiple potential phosphorylation sites.

The gene associated with CFTR has been identified and sequenced (See Gregory, RJ et al. (1990) Nature 347: 382386; Rich, DP et al. (1990) Nature 347: 358-362), (Riordan, JR et al. (1989) Science 245: 1066-1073). A defect in this gene leads to cystic fibrosis (hereinafter "CF"), the most common fatal genetic disease in humans, affecting approximately one in 2,500 children born in the United States. Within the general population of the United States, up to 10 million people carry a single copy of the defective gene with no apparent harmful effects. In contrast, individuals with two copies of the CF-associated gene suffer from the chronic effects of CF, including chronic lung destruction and death.

In CF patients, expression of the CF-associated gene in airway cells leads to reduced cellular apical chloride conductance that causes an imbalance in ion and fluid transport. Most believe that this leads to abnormal mucus secretion in the pancreatic ducts and airways, ultimately resulting in lung infections and epithelial cell damage typically associated with disease progression to CF. In addition to respiratory problems, CF patients typically suffer from gastrointestinal problems and pancreatic insufficiency. Men are almost uniformly infertile and fertility decreases in women. In contrast to the severe effects of possessing two copies of the CF-associated gene, individuals with a single copy of the CF-associated gene have increased resistance to cholera and dehydration resulting from diarrhea - perhaps explaining the relatively high frequency of this CF gene among the population.

Sequence analysis of the CFTR gene of CF chromosomes has revealed various diseases that cause mutations (Cutting, GR et al. (1990) Nature 346: 366-369; Dean, M. et al. (1990) Cell 61 : 863: 870; and Kerem, BS. Et al. (1989) Science 245: 1073-1080; Kerem, BS et al. (1990) Proc. Natl. Acad. Sci. USA 87: 84478451). Currently, more than 1000 mutations have been identified in the CF gene (http://www.genet.sickkids.on.ca/ cftr /), although demographic studies have indicated that the most common CF mutation, a deletion of the 3 nucleotides that encode phenylalanine at position 508 of the amino acid sequence of CFTR is associated with approximately 70% of cases of cystic fibrosis. The mutated CFTR protein is called AF508.

The deletion of residue 508 in AF508-CFTR is believed to prevent the nascent protein from folding correctly, resulting in the inability of this mutant protein to exit the endoplasmic reticulum (hereinafter "RE"), and target the plasma membrane. As a result, insufficient amounts of the mature protein are present in the plasma membrane and chloride transport within epithelial tissues is significantly reduced (Quinton, P. M. (1990), FASEB J. 4: 2709-2727). Therefore, the cellular phenomenon of defective ER processing into other proteins such as CFTR, by the ER machinery, has been shown to be the underlying basis for a wide range of isolated and inherited diseases. The two ways that

ES 2 328 824 T3 ER machinery may malfunction is by loss of coupling to ER export of proteins leading to degradation, or by accumulation in ER of these defective / misfolded proteins [AridorM, et al., Nature Med., 5 (7), p. 745-751 (1999); Shastry, BS, et al., Neurochem. International, 43, p. 1-7 (2003); Rutishauser, J., et al., Swiss Med Wkly, 132, p. 211-222 (2002); Morello, JP et al., TIPS, 21, p. 466-469 (2000); Brass P., et al., Human Mut, 14, p. 186-198 (1999)]. However, studies have shown that AF508RTFQ, when present in the plasma membrane, is functional as a Cl channel.^- sensitive to cAMP (Dalemans et al. (1991), Nature Lond. 354: 526-528; Denning et al., supra .; Pasyk and Foskett (1995), J. Cell. Biochem. 270: 1234750).

Although CFTR carries various molecules in addition to anions, this role of transporting anions represents an important element in the global cellular machinery for transporting ions and water through the epithelium. The other elements include the Na channel⁺ epithelial, ENaC, the Na co-transporter⁺/ 2Cl^-/ K⁺, the Na pump⁺-K⁺-ATPase and K channels⁺ basolateral membrane, which are sensitive to chloride uptake into the cell.

These elements work together to achieve directional transport through the epithelium through their expression and selective localization within the cell. Chloride absorption takes place through the coordinated activity of: (i) ENaC and CFTR present in the apical membrane; and (ii) the Na pump⁺ -K⁺-ATPase and Cl channels^- expressed on the basolateral surface of the cell. Active secondary chloride transport from the luminal side leads to intracellular chloride accumulation, which can then passively exit the cell through Cl channels.^-, resulting in a vector transport. The arrangement of the Na co-transporter⁺/ 2Cl^-/ K⁺, the Na pump⁺K⁺-ATPase and K channels⁺ of the basolateral membrane on the basolateral surface and CFTR on the luminal side coordinate chloride secretion by CFTR on the luminal side. Because water probably never actively transports itself, its flow through epithelia is dependent on small transepithelial osmotic gradients generated by the bulk of sodium and chloride flux.

In addition to CF, modulation of CFTR activity may be beneficial for other diseases not directly caused by CFTR mutations, such as secretory diseases and other diseases related to CFTR-mediated protein folding. These include, but are not limited to, chronic obstructive pulmonary disease (hereinafter "COPD"), dry eye disease, and Sjogrens syndrome.

COPD is characterized by airflow limitation that is progressive and not completely reversible. Airflow limitation is due to hypersecretion of mucus, emphysema, and bronchiolitis. Wild-type or mutant CFRT activators offer a potential treatment of the mucus hypersecretion and impaired mucociliary clearance that is common in COPD. Specifically, increasing anion secretion through CFTR can facilitate fluid transport into the superficial fluid of the airways to hydrate mucus and optimize periciliary fluid viscosity. This would lead to enhanced mucociliary clearance and a reduction in symptoms associated with COPD. Dry eye disease is characterized by a decrease in aqueous tear production and in the lipid, protein, and mucin profiles in the abnormal tear film. There are many causes of dry eye, some of which include Lasik eye surgery due to age, arthritis, medications, chemical / thermal burns, allergies, and illnesses, such as CF and Sjogrens syndrome. Increasing the secretion of anions by CFTR would enhance the transport of fluids from the corneal endothelial cells and the secretory glands that surround the eye to increase corneal hydration. This would help alleviate the symptoms associated with dry eye disease. Sjogrens syndrome is an autoimmune disease in which the immune system attacks moisture-producing glands throughout the body, including the eye, mouth, skin, respiratory tissue, liver, vagina, and intestine. Symptoms include dry eye, mouth, and vagina, as well as lung disease. The disease is also associated with rheumatoid arthritis, systemic lupus, systemic sclerosis, and polymyositis / dermatomyositis. Defective protein transport is believed to cause disease for which there are limited treatment options. Modulators of CFTR activity can hydrate the various organs affected by the disease and help to elevate the associated symptoms.

As discussed above, the deletion of residue 508 in AF508-CFTR is believed to prevent the nascent protein from folding correctly, resulting in the inability of this mutant protein to exit the ER, and target the plasma membrane. As a result, insufficient amounts of the mature protein are present in the plasma membrane and chloride transport within epithelial tissues is significantly reduced. In fact, it has been shown that this cellular phenomenon of defective ER processing in other proteins such as CFTR, by the ER machinery, is the underlying basis not only for CF disease, but for a wide range of other isolated diseases and hereditary. The two ways in which the ER machinery can malfunction are through loss of coupling to ER export from proteins leading to degradation, or through ER accumulation of these defective / misfolded proteins [Aridor M, et al. ., Nature Med., 5 (7), p. 745-751 (1999); Shastry, BS, et al., Neurochem. International, 43, p. 1-7 (2003); Rutishauser, J., et al., Swiss Med Wkly, 132, p. 211-222 (2002); Morello, JP et al., TIPS, 21, p. 466-469 (2000); Brass P., et al., Human Mut, 14, p. 186-198 (1999)].

Diseases associated with the first class of ER malfunction are CF (due to AF508RTFQ being misfolded), hereditary emphysema (due to α1-antitrypsin; without Piz variants), hereditary homochromatosis, coagulation-fibrinolysis deficiencies, such as protein C deficiency, Type 1 hereditary angioedema, lipid processing deficiencies, such as familial hypercholesterolemia, chylomicronemia Type 1, abetalipoproteinemia, lysosomal storage diseases, such as I cell / pseudo-Hurler disease,

ES 2 328 824 T3 mucopolysaccharidoses (due to lysosomal processing enzymes), Sandhof / Tay-Sachs (due to aj6-hexosaminidase), Crigler-Najjar type II (due to UDP-glucuronyl-sialic-transferase), poliendocrinopathy / hyperinsulemia, Diabetes mellitus (due to insulin receptor), Laron's dwarfism (due to growth hormone receptor), myeloperoxidase deficiency, primary hypoparathyroidism (due to preproparathyroid hormone), melanoma (due to tyrosinase). The diseases associated with this last class of ER malfunction are CDG type 1 glucanosis, hereditary emphysema (due to a1-antitrypsin (PiZ variant), congenital hyperthyroidism, osteogenesis imperfecta (due to type I, II, IV procollagen), Hereditary hypofibrinogenemia (due to fibrinogen), ACT deficiency (due to α1-antichymotrypsin), Diabetes insipidus (DI), Neurophyseal DI (due to vasopressin hormone / V2 receptor), Neprogenic DI (due to aquaporin II), Charcot-Marie Tooth syndrome (due to peripheral myelin protein 22), Perlizaeus-Merzbacher disease, neurodegenerative diseases such as Alzheimer's disease (due to q'APP and presenilins), Parkinson's disease, amyotrophic lateral sclerosis, progressive supranuclear palsy , Pick's disease, various polyglutamine-related neurological disorders such as Huntington's spinocerebellar ataxia type I, spinal and bulbar muscular atrophy, dentatorubal pallidoluisiana, and myotonic dystrophy, as well as spongiform encephalopathies, such as inherited Creutzfeldt-Jakob disease (due to a prion protein processing defect), Fabry disease (due to lysosomal α-galactosidase A) and Straussler syndrome- Scheinker (due to a Prp processing defect).

In CF, CFTR-mediated chloride transport is reduced resulting in abnormal mucus secretion that characterizes the disease. In contrast, in secretory diarrhea epithelial water transport increases dramatically as a result of chloride transport activated by secretagogues. The mechanism involves elevation of cAMP and stimulation of CFTR.

Although there are numerous causes of diarrhea, the main consequences of diarrheal diseases resulting from excessive chloride transport are common to all, and include dehydration, acidosis, death, and reduced growth.

Acute and chronic diarrhea represent a major medical problem in many areas of the world. Diarrhea is both a significant factor in malnutrition and the leading cause of death (5,000,000 deaths / year) in children under five years of age.

Secretory diarrhea is also a dangerous condition in patients with acquired immunodeficiency syndrome (AIDS) and chronic inflammatory bowel disease (IBD). Sixteen million travelers to developing countries from industrialized nations each year develop diarrhea, with the severity and number of diarrhea cases varying depending on the country and area visited.

Diarrhea in livestock and companion animals such as cows, pigs and horses, sheep, goats, cats and dogs, also known as purging, is a leading cause of death in these animals. Diarrhea can be the result of any major transition, such as weaning or physical movement, as well as in response to various bacterial or viral infections and generally occurs during the first hours of the animal's life.

The most common diarrhea-causing bacteria is enterotoxogenic E-coli (ETEC) which has the hairy K99 antigen. Common viral causes of diarrhea include rotavirus and coronavirus. Other infectious agents include cryptosporidium, giardia lamblia, and salmonella, among others.

Symptoms of rotaviral infection include watery stool, dehydration, and weakness. The coronavirus causes more serious disease in newborn animals, and has a higher mortality rate than rotaviral infection. However, often a young animal can become infected with more than one virus or with a combination of viral and bacterial microorganisms at the same time. This dramatically increases the severity of the disease.

Accordingly, there is a need for modulators of ABC transporter activity, and compositions thereof, that can be used to modulate ABC transporter activity in the cell membrane of a mammal.

There is a need for methods of treating ABC transporter mediated diseases using such modulators of ABC transporter activity.

There is a need for methods to modulate an ABC transporter activity in a mammalian ex vivo cell membrane.

There is a need for modulators of CFTR activity that can be used to modulate CFTR activity in the cell membrane of a mammal.

There is a need for methods of treating CFTR-mediated diseases using such modulators of CFTR activity.

There is a need for methods to modulate CFTR activity in an ex vivo cell membrane of a mammal. Agric. Chem. Biotechnol. 45 (1) 37-42 (2002) describes 1,3-trifluoromethylated oxazolecarbonanilides and their use as anti-fungal compositions. EP 0 652 229 A2 describes oligopeptide compounds having antifungal activity. EP 0 419 944 A2 describes oxazole or thiazole carboxylic acid amides which

ES 2 328 824 T3 have herbicidal activity. GB 1 318291 describes a 2,5-dimethylthiazole-3- (N- (3,4-methylenedioxyphenyl) carboxamide with anti-fungal activity for the purpose of protecting crops. US 6 630 482 B1 describes activating compounds of the CFTR channel WO 2004/084842 A2 describes cathepsin S inhibitors. DE 102 50 110 A1 describes thiazole- (bi) cycloalkyl carboxanilides for the elimination of unwanted microorganisms.

Summary of the invention

The compounds used in the present invention, and their pharmaceutically acceptable compositions, have now been found to be useful as modulators of ABC transporter activity. These compounds have the general formula I:

<img file="ES2328824T3_D0001.tif" />

or its pharmaceutically acceptable salt, where X, Z, R¹, R², R³, and R⁴ they are described generally and in classes and subclasses below.

These compounds and their pharmaceutically acceptable compositions are useful for treating or lessening the severity of various diseases, disorders, or conditions including, but not limited to, cystic fibrosis, hereditary emphysema, hereditary homochromatosis, coagulation-cyibrinolysis deficiencies, such as protein C deficiency. , Type 1 hereditary angioedema, lipid processing deficiencies such as familial hypercholesterolemia, Type 1 chylomicronemia, abetalipoproteinemia, lysosomal storage diseases, such as I cell / pseudo-Hurler disease, secretory diarrhea or polycystic kidney disease, mucopolysaccharidoses, Sandhof / Tay-Sachs, Crigler-Najjar type II, poliendocrinopathy / hyperinsulemia, Diabetes mellitus, Laron's dwarfism, myeloperoxidase deficiency, primary hypoparathyroidism, melanoma, CDG type 1 glucanosis, hereditary emphysema, congenital hyperthyroidism, osteogenesis imperfecta, Hereditary hypofibrinogenemia, ACT deficiency, Diabetes insipidus (DI), neurophyseal DI, neprogenic DI, Charcot-Marie Tooth syndrome, Pelizaeus-Merzbacher disease, neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, supranuclear palsy progressive, Pick's disease, various polyglutamine-related neurological disorders such as Huntington's spinocerebellar ataxia type I, Spinal and bulbar muscular atrophy, dentatorubal pallidoluisian, and myotonic dystrophy, as well as spongiform encephalopathies, such as inherited Creutzfeldt-Jakob disease (due to a cryonic protein processing defect), Fabry disease, Straussler-Scheinker syndrome, COPD , dry eye disease or Sjogren's disease.

Detailed description of the invention

1. General Description of the Compounds of the Invention

The present invention relates to compounds of formula I useful as modulators of ABC transporter activity:

<img file="ES2328824T3_D0002.tif" />

or its pharmaceutically acceptable salt, where X is O or S;

Z is a bond or is a C alkylidene chain_1-4 optionally substituted, wherein up to two methylene units in the chain are optionally and independently substituted with -NR-, -S-, -O-, -SO2NR-, -NRSO2-, -SO2-, or -CO-;

ES 2 328 824 T3

R¹ is hydrogen, or is a saturated, partially unsaturated, or fully unsaturated 3-8 membered monocyclic ring having 0-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or a saturated 8-12 membered bicyclic ring system, partially unsaturated, or fully unsaturated having 0-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur, where R¹ is optionally substituted on one or more carbon or nitrogen atoms with x independent occurrences of -QRX; where x is 05; where Q is a bond or is a C alkylidene chain₁-C₆ wherein up to two methylene units of Q are optionally and independently substituted with -CO-, -CO₂-, -COCO-, -CONR-, -OCONR-, -NRNR-, -NRNRCO-, -NRCO-, -NRCO2-, -NRCONR-, -SO-, -SO2-, -NRSO2-, -SO2NR-, -NRSO2NR-, -O-, -S-; or -NR-; and each occurrence of RX is independently R ', halogen, NO2, or CN, or -QRX is = O, = S, or = NR';

each occurrence of R is independently hydrogen or an aliphatic group C_1-6 optionally substituted; and each occurrence of R 'is independently hydrogen or an optionally substituted C1-C6 aliphatic group, a 3-8 membered saturated, partially unsaturated, or fully unsaturated monocyclic ring having 0-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur. , or a saturated, partially unsaturated, or fully unsaturated 8-12 membered bicyclic ring system having 0-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur; or R and R ', two occurrences of R, or two occurrences of R', are taken together with the atom or atoms to which they are attached to form a saturated, partially unsaturated, or fully 3-12 membered monocyclic or bicyclic ring unsaturated, optionally substituted, having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur;

R² is halogen, -CN, -CF3, -NO2, or -TqR;

R³ is U_mR 'and R⁴ is VpCy¹, wherein each m, p, and q is independently 0 or 1, and each U, V, and T is independently a C alkylidene chain₁-C₄ optionally substituted, wherein up to two methylene units in the chain are optionally and independently substituted with -CO-, -CO2-, -COCO-, -CONR-, -OCONR-, -NRNR-, -NRNRCO-, -NRCO- , -NRCO2-, -NRCONR-, -SO-, -SO2-, -NRSO2-, -SO2NR-, -NRSO2NR-, -O-, -S-; or -NR-;

Cy¹ is a saturated, partially unsaturated, or fully unsaturated 3-8 membered monocyclic ring having 0-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or a saturated, partially unsaturated, or 8-12 membered bicyclic ring system fully unsaturated having 0-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur, where Cy¹ is optionally substituted on one or more carbon or nitrogen atoms with, and independent occurrences of, -WR^W; where y is 0-5; wherein W is a bond or is a C1-C6 alkylidene chain in which up to two methylene units of W are optionally and independently substituted with -CO-, -CO2-, -COCO-, -CONR-, -OCONR- , -NRNR-, -NRNRCO-, -NRCO-, -NRCO2-, -NRCONR-, -SO-, -SO2-, -NRSO2-, -SO2NR-, -NRSO2NR-, -O-, -S-; or -NR-; and every occurrence of R^W is independently R ', halogen, NO₂, or CN, or -WR^W is = O, = S, or = NR '; or

R³ and R⁴, taken together with the nitrogen atom to which they are attached, form a saturated, partially unsaturated or fully unsaturated, optionally substituted 5, 6 or 7 membered ring, having the structure:

<img file="ES2328824T3_D0003.tif" />

<img file="ES2328824T3_D0004.tif" />

<img file="ES2328824T3_D0005.tif" />

where r is 0, 1, or 2; one of X³, X⁴, or X⁵ is CH-Vp-Cy¹ or N-Vp-Cy¹, and the others of X³, X⁴, or X⁵ are CHR 'or NR'; and every occurrence of X¹, when present, and X² is each independently C (R ')₂, -CO-, or -CS-.

In some embodiments, for compounds of formula I as generally described above:

a) when X is S, Z is CH₂, and R² is hydrogen, so R¹ It is not:

<img file="ES2328824T3_D0006.tif" />

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b) when X is S or O; and R² is formyl, 4,5-dihydro-2-oxazolyl, -COOR ', or -COSR'; then:

i) when Z is -CH₂-, R¹ not phenyl optionally substituted with C alkyl₁-C₄, haloalkyl C₁ -C₄, Ci-C alkoxy₄, haloalkoxy C₁-C₄, C, -C alkyl₄ thio, haloalkyl C, -C₄ thio, halogen, cyano, or nitro; or ii) when Z is C alkyl_1-6, then R¹ not cycloalkyl C₃-C₆ optionally substituted with C, -C alkyl₄, haloalkyl C₁-C₄, C alkoxy₁-C₄, haloalkoxy C₁-C₄, C alkyl₁-C₄ thio, haloalkyl C₁-C₄ thio, halogen, cyano, or nitro;

c) when X is S, R² is H, Z is -CH₂-, and R¹ is unsubstituted phenyl, then when R³ is hydrogen, R⁴ It is not:

<img file="ES2328824T3_D0007.tif" />

d) when X is S, R² is SMe or Me, Z is CH₂ and R¹ is unsubstituted phenyl.

i) when R³ is hydrogen, so R⁴ it is not unsubstituted benzyl; and ii) when R³ is ethyl, so R⁴ it is not unsubstituted phenyl;

e) when X is O, Z is CH₂, R¹ is unsubstituted phenyl, R² is Me, and R³ is hydrogen, so R⁴ It is not

<img file="ES2328824T3_D0008.tif" />

f) when X is O, Z is CH₂, R¹ is unsubstituted phenyl, R² is hydrogen, and R³ is hydrogen, so R⁴ it is not unsubstituted phenyl, 4-Me-phenyl, or unsubstituted benzyl; Y

g) when X is O, Z is -CH (Tr) -, R¹ it is

<img file="ES2328824T3_D0009.tif" />

and R³ is hydrogen, so R⁴ is not - (CH₂)₂-S-Phenyl;

h) when ZR¹ is methyl, X is S, R³ is iBu, so R⁴ It is not:

<img file="ES2328824T3_D0010.tif" />

i) when ZR¹ is phenyl, X is S, and R₃ is - (CH₂)₂-pyrid-2-yl, then R⁴ It is not

<img file="ES2328824T3_D0011.tif" />

ES 2 328 824 T3

2. Compounds and Definitions

Compounds used in the present invention include those generally described above, and are further illustrated by the classes, subclasses, and species described herein. As used herein, the following definitions apply unless otherwise indicated. For the purposes of the present invention, chemical elements are identified according to the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75^to Ed. In addition, the general principles of organic chemistry are described in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 1999, and "March's Advanced Organic Chemistry", 5th Ed., Ed .: Smith, MB and March , J., John Wiley &amp; Sons, New York: 2001, the full contents of which are incorporated herein by reference.

As described herein, the compounds used in the invention may be optionally substituted with one or more substituents, such as those generally illustrated above, or as exemplified by the particular classes, subclasses, and species of the invention. It will be appreciated that the term "optionally substituted" is used interchangeably with the term "substituted or unsubstituted." In general, the term "substituted", whether or not preceded by the term "optionally", refers to the substitution of hydrogen radicals in a given structure with the radical of a specified substituent. Unless otherwise indicated, an optionally substituted group may have a substituent at each substitutable position in the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be the same or different in each position. The combinations of substituents envisioned by the present invention are preferably those that result in the formation of stable or chemically feasible compounds. The term "stable", as used herein, refers to compounds that are not substantially altered when subjected to conditions that allow their production, detection, and preferably recovery, purification, and use for one or more of the purposes described in this document. In some embodiments, a stable compound or a chemically feasible compound is one that is not substantially altered when held at a temperature of 40 ° C or lower, in the absence of moisture, light, or other chemically reactive conditions, for at least one week. .

The term "aliphatic" or "aliphatic group", as used herein, refers to a substituted or unsubstituted hydrocarbon chain, straight chain (ie, unbranched) or branched, that is fully saturated or that contains one or more units of unsaturation, or a monocyclic hydrocarbon or bicyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic (also referred to herein as "carbocycle", "cycloaliphatic" or "cycloalkyl"), having a single point of attachment to the rest of the molecule. Unless otherwise specified, aliphatic groups contain 1-20 aliphatic carbon atoms. In some embodiments, the aliphatic groups contain 1-10 aliphatic carbon atoms. In other embodiments, the aliphatic groups contain 1-8 aliphatic carbon atoms. In still other embodiments, the aliphatic groups contain 1-6 aliphatic carbon atoms, and in still other embodiments the aliphatic groups contain 1-4 aliphatic carbon atoms. In some embodiments, "cycloaliphatic" (or "carbocycle" or "cycloalkyl") refers to a monocyclic hydrocarbon C₃-C₈ or bicyclic hydrocarbon C₈-C₁₂ that is fully saturated or that contains one or more units of unsaturation, but that is not aromatic, that has a single point of attachment to the rest of the molecule in which any single ring in said bicyclic ring system has 3-7 members. Suitable aliphatic groups include, but are not limited to, alkyl, alkenyl, alkynyl, substituted or unsubstituted, linear or branched, and hybrids thereof such as (cycloalkyl) alkyl, (cycloalkenyl) alkyl, or (cycloalkyl) alkenyl.

The term "heteroaliphatic", as used herein, refers to aliphatic groups in which one or two carbon atoms are independently substituted with one or more of oxygen, sulfur, nitrogen, phosphorus, or silicon. Heteroaliphatic groups may be substituted or unsubstituted, or branched or unbranched, cyclic or acyclic, and include "heterocycle", "heterocyclyl", "heterocycloaliphatic", or "heterocyclic" groups.

The term "heterocycle", "heterocyclyl", "heterocycloaliphatic", or "heterocyclic" as used herein refers to non-aromatic, monocyclic, bicyclic, or tricyclic ring systems in which one or more ring members are an independently selected heteroatom. In some embodiments, the "heterocycle", "heterocyclyl", "heterocycloaliphatic", or "heterocyclic" group has three to fourteen ring members in which one or more ring members is a heteroatom independently selected from oxygen, sulfur, nitrogen, or phosphorus, and each ring in the system contains 3 to 7 ring members.

The term "heteroatom" refers to one or more of oxygen, sulfur, nitrogen, phosphorus, or silicon (including, any oxidized form of nitrogen, sulfur, phosphorus, or silicon; the quaternized form of any basic nitrogen or; a substitutable nitrogen of a heterocyclic ring, for example N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) or NR⁺ (as in N-substituted pyrrolidinyl)).

The term "unsaturated", as used herein, means that a moiety has one or more units of unsaturation.

The term "alkoxy" or "thioalkyl", as used herein, refers to an alkyl group, as defined above, attached to the main carbon chain via an oxygen ("alkoxy") or sulfur atom. ("Thioalkyl").

ES 2 328 824 T3

The expressions "haloalkyl", "haloalkenyl" and "haloalkoxy" refer to alkyl, alkenyl or alkoxy, as the case may be, substituted with one or more halogen atoms. The term "halogen" refers to F, Cl, Br, or I.

The term "aryl" used alone or as part of a larger moiety such as "aralkyl", "aralkoxy", or "aryloxyalkyl", refers to monocyclic, bicyclic, and tricyclic ring systems that have a total of five to fourteen ring members, in which at least one ring in the system is aromatic and in which each ring in the system contains 3 to 7 ring members. The term "aryl" can be used interchangeably with the term "aryl ring". The term "aryl" also refers to heteroaryl ring systems as defined herein below.

The term "heteroaryl", used alone or as part of a larger moiety as in "heteroaralkyl" or "heteroarylalkoxy", refers to monocyclic, bicyclic, and tricyclic ring systems having a total of five to fourteen members in the ring, where at least one ring in the system is aromatic, at least one ring in the system contains one or more heteroatoms, and where each ring in the system contains 3 to 7 ring members. The term "heteroaryl" can be used interchangeably with the term "heteroaryl ring" or the term "heteroaromatic."

An aryl (including aralkyl, aralkoxy, aryloxyalkyl, and the like) or heteroaryl (including heteroaralkyl and heteroarylalkoxy, and the like) group may contain one or more substituents, and thus may be "optionally substituted." Unless otherwise defined above and herein, suitable substituents on the unsaturated carbon atom of an aryl or heteroaryl group are generally selected from halogen; -R °; -OR °; -SR °; phenyl (Ph) optionally substituted with R °; -O (Ph) optionally substituted with R °; - (CH₂) i-₂(Ph), optionally substituted with R °; -CH = CH (Ph), optionally substituted with R °; -DO NOT₂; -CN; -N (R °)₂; -NR ° C (O) R °; -NR ° C (S) R °; -NR ° C (O) N (R °)₂; -NR ° C (S) N (R °)₂; -NR ° CO₂R °; -NR ° NR ° C (O) R °; -NR ° NR ° C (O) N (R °) 2; -NR ° NR ° CO2R °; -C (O) C (O) R °; -C (O) CH2C (O) R °; -CO2R °; -C (O) R °; -C (S) R °; -C (O) N (R °) 2; -C (S) N (R °) 2; -OC (O) N (R °) 2; -OC (O) R °; -C (O) N (OR °) R °; -C (NOR °) R °; -S (O) 2R °; -SW)₃R °; -SQ¡N (R^or)2; -S (O) R °; -NR ° SO2N (R °) 2; -NR ° SO2R °; -N (OR °) R °; -C (= NH) -N (R °) 2; -P (O) 2R °; -PO (R °) 2; -OPO (R ° b; - (CH ·) -XHCcOíRO; phenyl (Ph) optionally substituted with R °; -O (Ph) optionally substituted with R °; - (CH₂)_1-2(Ph), optionally substituted with R °; or -CH = CH (Ph), optionally substituted with R °; in which each independent occurrence of R ° is selected from hydrogen, aliphatic C_1-6 optionally substituted, an unsubstituted 56-membered heteroaryl or heterocyclic ring, phenyl, -O (Ph), or -CH₂(Ph), or, notwithstanding the above definition, two independent occurrences of R °, in the same substituent or in different substituents, taken together with the atom or atoms to which each R ° group is attached, to form a monocyclic ring or bicyclic, saturated, 3-12 membered, partially unsaturated, or fully unsaturated, optionally substituted, having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

Optional substituents on the aliphatic group of R ° are selected from NH₂, NH (aliphatic C_1-4), N (aliphatic C_1-4)₂, halogen, aliphatic C_1-4, OH, O (aliphatic C_1-4), DO NOT₂, CN, CO₂H, CO₂(aliphatic C_1-4), O (haloaliphatic C_1-4), or haloaliphatic C_1-4, in which each of the aliphatic groups C_1-4 above of R ° is not substituted.

An aliphatic or heteroaliphatic group, or a non-aromatic heterocyclic ring may contain one or more substituents and thus may be "optionally substituted". Unless otherwise defined above and herein, suitable substituents on the saturated carbon of an aliphatic or heteroaliphatic group, or a non-aromatic heterocyclic ring are selected from those listed above for the unsaturated carbon of a group aryl or heteroaryl and additionally include the following: = O, = S, = NNHR *, = NN (R *)₂, = NNHC (O) R *, = NNHCO₂(alkyl), = NNhSo₂(alkyl), o = NR *, where each R * is independently selected from hydrogen or an aliphatic group C_1-6 optionally substituted.

Unless defined otherwise above and herein, optional substituents on the nitrogen of a non-aromatic heterocyclic ring are generally selected from -R⁺, -N (R⁺) 2, -C (O) R⁺, -CO2R⁺, -C (O) C (O) R⁺, -C (O) CH2C (O) R⁺, -SO2R⁺, -SO2N (R⁺) 2, -C (= S) N (R⁺¹) 2, -C (= NH) -N (R⁺) 2, or -NR⁺SO2R⁺; in which R⁺ is hydrogen, optionally substituted C1-6 aliphatic, optionally substituted phenyl, optionally substituted -O (Ph), -CH₂(Ph) optionally substituted, - (CH₂)_1-2(Ph) optionally substituted; -CH = CH (Ph) optionally substituted; or a 5-6 membered, unsubstituted heteroaryl or heterocyclic ring having one to four heteroatoms independently selected from oxygen, nitrogen, or sulfur, or, notwithstanding the above definition, two independent occurrences of R⁺, in the same substituent or in different substituents, taken together with the atom or atoms to which each R group⁺ is bonded to form a 3-12 membered saturated, partially unsaturated, or fully unsaturated, optionally substituted monocyclic or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

Optional substituents on the aliphatic group or phenyl ring of R⁺ are selected from -NH₂, -NH (aliphatic C_1-4), -N (aliphatic C_1-4)₂, halogen, aliphatic C_1-4, -OH, -O (aliphatic C_1-4), -DO NOT₂, -CN, -CO₂H, -CO₂(aliphatic C_1-4), -O (aliphatic halo C_1-4), or halo (aliphatic C_1-4), in which each of the aliphatic groups C_1-4 previous R⁺ is not substituted.

The term "alkylidene chain" refers to a straight or branched carbon chain that can be fully saturated or have one or more units of unsaturation and has two points of attachment to the rest of the molecule.

ES 2 328 824 T3

As detailed above, in some embodiments, two independent occurrences of R ° (or R⁺, R, R 'or any other variable defined similarly herein), are taken together with the atom or atoms to which they are attached to form a saturated, partially unsaturated, or 3-12 membered monocyclic or bicyclic ring fully unsaturated, optionally substituted, having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

The exemplary rings that are formed when two independent occurrences of R ° (or R⁺, R, R 'or any other variable defined similarly herein), taken together with the atom or atoms to which each variable is attached include, but are not limited to, the following: a) two independent occurrences of R ° (or R⁺, R, R 'or any other variable defined similarly herein) that are attached to the same atom and taken together with that atom to form a ring, for example, N (R °)₂, where both occurrences of R ° are taken together with the nitrogen atom to form a piperidin-1-yl, piperazin-1-yl, or morpholin-4-yl group; and b) two independent occurrences of R ° (or R⁺, R, R 'or any other variable similarly defined herein) that are attached to different atoms and taken together with both of these atoms to form a ring, for example where one phenyl group is substituted with two OR ° appearances

<img file="ES2328824T3_D0012.tif" />

These two occurrences of R ° are taken together with the oxygen atoms to which they are attached to form a 6-membered condensed ring containing oxygen:

<img file="ES2328824T3_D0013.tif" />

It will be appreciated that various other rings can be formed when two independent occurrences of R ° (or R⁺, R, R 'or any other variable similarly defined herein) are taken in conjunction with the atom or atoms to which each variable is attached and that the examples detailed above are not intended to be limiting.

Unless otherwise indicated, the structures depicted herein are intended to also include all isomeric (eg, enantiomeric, diastereomeric, and geometric (or conformational) forms) of the structure; for example, the R and S configurations for each asymmetric center, double bond isomers (Z) and (E), and conformational isomers (Z) and (E). Therefore, individual stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the compounds of the present invention are within the scope of the invention. Unless otherwise indicated, all tautomeric forms of the compounds of the invention are within the scope of the invention. Furthermore, unless otherwise indicated, the structures depicted herein are also intended to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures except for the substitution of hydrogen for deuterium or tritium, or the substitution of a carbon for a carbon enriched in¹³C or ¹⁴C are within the scope of the present invention. Such compounds are useful, for example, as analytical tools or probes in biological assays.

The term "ABC transporter" as used herein refers to an ABC transporter protein or a fragment thereof comprising at least one binding domain, wherein said protein or fragment thereof is present in vivo or in vitro. The term "binding domain" as used herein refers to a domain on the ABC transporter that can bind to a modulator. See, for example, Hwang, TC et al., J. Gen. Physiol. (1998): 111 (3), 477-90.

The term "CFTR" as used herein refers to the cystic fibrosis transmembrane conductance regulator or a mutation thereof capable of regulatory activity, including, but not limited to, AF508 CFTR and G551D CFTR (see, for example , http: //www.genet.sickkids.on.ca/cftr/, for CFTR mutations).

The term "modulate" as used herein refers to increasing or decreasing by a measurable amount.

ES 2 328 824 T3

3. Description of Exemplary Compounds

As generally described above, for the compounds used in the invention, X is O or S and compounds of formula IA or IB are provided:

<img file="ES2328824T3_D0014.tif" />

As generally described above, Z is an optionally substituted C 1-4 alkylidene chain or linkage, wherein up to two methylene units in the chain are optionally and independently substituted with -NR-, -S-, -O -, or -CO-. In certain exemplary embodiments, Z is - [C (R⁵) 2] n-, where n is 0, 1, 2, or 3, and each occurrence of R⁵ is independently halogen, CN, NO2, or -YR, where Y is a bond or is a C alkylidene chain₁C4 optionally substituted wherein up to two methylene units are optionally and independently substituted with -O-, -NR-, -CO-, -S-, -SO-, or -SO₂-. In other exemplary embodiments Z is - [C (R⁵) 2] nO-, where n is 0, 1, 2, or 3, and each occurrence of R⁵ is independently halogen, CN, NO2, or -YR, where Y is a bond or is a C1-C alkylidene chain₄ optionally substituted wherein up to two methylene units of Y are optionally and independently substituted with -O-, -NR-, -CO-, -S-, -SO-, or -SO₂-. In other exemplary embodiments plus Z is - [C (R⁵) 2] nS-, where n is 1, 2, or 3, and each occurrence of R⁵ is independently halogen, CN, No2, or -YR, where it is a bond or chain of C alkylidene₁ -C₄ optionally substituted wherein up to two methylene units of Y are optionally and independently substituted with -O-, -NR-, -CO-, -S-, -SO-, or -SO2-.

In some other embodiments, Z is a bond. In still other embodiments, Z is -C (R⁵)2-.

In certain embodiments, Z is -CH2- or -CH2-CH2-. In some embodiments, Z is -CH2-.

As generally described above, R¹ is hydrogen, or is a saturated, partially unsaturated, or fully unsaturated 3-8 membered monocyclic ring having 0-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or a saturated 8-12 membered bicyclic ring system, partially unsaturated, or fully unsaturated having 0-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur, where R¹ is optionally substituted on one or more carbon atoms with x independent occurrences of -QRX; where x is 0-5. In some embodiments R¹ it is hydrogen. In other embodiments R¹ is selected from one of the following groups:

<img file="ES2328824T3_D0015.tif" />

ES 2 328 824 T3

<td>H 4 N '^Nx í-.íü h s xCR³^)</td><td>* («* $&gt; i-iS «“ -N H</td>

<td>i</td><td>* J</td>

<td>V</td><td> %?</td>

<td>m</td><td>n</td>

<td>Qr^XN ** C IL&gt;^N</td><td>cw^xO ^_N·&lt;</td>

<td>what</td><td>r</td>

<td>Qr^Xh | A ^</td><td>0-4 V&gt; “(QR * ^</td>

<td>OR</td><td></td>

¢ -1 - ^. (0 ^), fl-J ^ ICWO,

V k 1

<td> 1 ^xs or</td><td>QR * tr4 JL '^NX /⁰P</td>

<td>Qr^X</td><td>Qr^X</td>

<td>rS » 1 /</td><td>nA&gt;</td>

<td>^ / = N</td><td> 4»</td>

<td> $</td><td>t</td>

<td>μΠ rt ^ J _w</td><td>| r ^ NH *</td>

4- (wr^w) „| 4 jH^qrX&gt; x

<td>Y</td><td>z</td>

<td>H i -&gt; (OR^X)_X</td><td>. ^ - &lt;OR^xk</td>

<td>DC</td><td>dd</td>

<td>K? ^ (QR ^ x aa</td><td>ym&gt; (QRX)_xbaby</td>

<td>_χ\ Onqr^x)_x</td><td></td>

ee

ES 2 328 824 T3

<img file="ES2328824T3_D0016.tif" />

In some embodiments R¹ is one of rings a, b, c, d, m, n, o, ee, gg, or pp. In other embodiments R¹ is ring a. In some other embodiments, R¹ is the gg ring.

It will be appreciated that, as generally described above, R¹ is optionally substituted on one or more carbon or nitrogen atoms with x independent occurrences of -QRX; where x is 0-5; where Q is a bond or is a C alkylidene chain₁ -C₆ wherein up to two methylene units of Q are optionally and independently substituted with -CO-, -CO₂-, -COCO-, -CONR-, -OCONR-, -NRNR-, -NRNRCO-, -NRCO-, -NRCO₂-, -NRCONR-, -SO-, -SO2-, -NRSO2-, -SO2NR-, -NRSO2NR-, -O-, -S-; or -NR-; and each occurrence of RX is independently R ', halogen, NO2, or CN, or -QRX is = O, = S, or = NR'. In certain embodiments, x is 0, 1, 2, or 3, and Q is a bond or is a C alkylidene chain₁ -C₆ optionally substituted wherein one or two methylene units are optionally and independently substituted with O, NR, S, SO2, or CO2, CO, and RX is R 'or halogen. In other embodiments, x is 0, 1, 2, or 3 and each occurrence of QRX, when present, is independently -C 1-3 alkyl, - ((C1_alkyl₃), -CF3, -OCF3, -SCF3, -F, -Cl, -Br, -COOR ', -COR', -O (CH₂) 2N (R) (R '), -O (CH2) N (R) (R'), -CON (R) (R '), - (CH₂)₂OR ', - (CH₂) OR ', optionally substituted phenyl, optionally substituted benzyl, -N (R) (R'), - (CH2) 2N (R) (R '), - (CH2) N (R) (R') -, or SO2NRR '.

As generally described above, R² it is halogen, -CF3, -CN, -NO2, or -TqR. In certain embodiments, R² is TqR, where T is a bond or is a C1-C alkylidene chain₄, wherein one or two methylene units of T are optionally substituted with -CO-, -CONR-, -SO₂-, -NRSO₂-, -SO₂NR-, -O-, -S-, or -NR. In other preferred embodiments, R² it is hydrogen or is optionally substituted C1-C4 alkyl. In still other preferred embodiments, R² is hydrogen, -CF3, -CH3, -CH2CH3, -CH2CH2CH3, -CH2OR - (CllH) R, - (CH) .OR, -CH2N (R) 2, - (CH2) 2N (R) 2, - (CH2)₃N (R) 2, -CH2NRCOR - (CH2ENRCOR, or- (CH2) sNRCOR.

ES 2 328 824 T3

As generally described above, R³ is U_mR 'and R⁴ is VpCy¹; where m, p, U, V, and Cy¹ have been defined generally above and herein; or R³ and R⁴, taken together with the nitrogen atom to which they are attached, form a saturated, partially unsaturated or fully unsaturated, optionally substituted 5, 6 or 7 membered ring, having the structure:

<img file="ES2328824T3_D0017.tif" />

where r is 0, 1, or 2; one of X³, X⁴, or X⁵ is CH-Vp-Cy¹ or N-Vp-Cy¹, and the others of X³, X⁴, or X⁵ are CHR 'or NR'; every occurrence of X¹, when present, and X² is each independently C (R ')₂, -CO-, or -CS-.

In certain embodiments for compounds of general formulas I, IA, or IB, R³ is UmR 'and R⁴ is VpCy¹. In some embodiments, R³ it is hydrogen. In still other embodiments, R³ is optionally substituted C1-4 alkyl. In other more R³ is UmR 'where m is 1 and U is -CH2- and R' is an optionally substituted saturated, partially unsaturated, or fully unsaturated 5- or 6-membered ring having 0-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In certain exemplary embodiments, R 'is an optionally substituted phenyl or pyridyl group. In some embodiments, p is 1 and V is -CH2C (R⁶) 2- or -C (R⁶) 2-, in which each occurrence of R⁶ is independently halogen, CN, NO2, or -YR, where Y is a bond or is a C alkylidene chain₁-C₄optionally substituted wherein up to two methylene units of Y are optionally and independently substituted with -O-, -NR-, -CO-, -S-, -SO-, or -SO2-, or wherein both occurrences of R⁶ on the same carbon atom are taken together to form an optionally substituted 3-6 membered spiro ring having 0-3 heteroatoms. In certain embodiments, R⁶ is hydrogen, methyl, or two occurrences of R⁶ on the same carbon atom they are taken together to form an optionally substituted 3-6 membered spiro ring having 0, 1 or 2 heteroatoms selected from nitrogen, oxygen, or sulfur. In still other embodiments, p is 0 and R⁴ is Cy¹.

In still other embodiments, R³ and R⁴, taken together with the nitrogen atom to which they are attached, form a saturated, partially unsaturated or fully unsaturated, optionally substituted 5, 6 or 7 membered ring, having the structure:

<img file="ES2328824T3_D0018.tif" />

where r is 0, 1, or 2; one of X³, X⁴, or X⁵ is CH-Vp-Cy¹ or N-Vp-Cy¹, and the others of X³, X⁴, or X⁵ are CHR 'or NR'; every occurrence of X¹, when present, and X² is each independently C (R ') 2, -CO-, or -CS-. In certain embodiments, r is 1, each X¹, X², X³ and X⁵ is CH2, and X⁴ is CH-V -Cy1, or NV -Cy¹. In some other embodiments, p is 1 and V is SO2, -NRSO₂, CO, or NRCO. In still other embodiments, X⁴ is N-Vp-Cy¹, p is 1 and V is SO2 or CO.

As generally described above, Cy¹ is a saturated, partially unsaturated, or fully unsaturated 3-8 membered monocyclic ring having 0-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or a saturated, partially unsaturated, or 8-12 membered bicyclic ring system fully unsaturated having 0-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur, where Cy¹ is optionally substituted on one or more carbon or nitrogen atoms with, and independent occurrences of, -WR^W; where y is 0-5; wherein W is a bond or is a C1-C6 alkylidene chain in which up to two methylene units of W are optionally and independently substituted with -CO-, -CO2-, -COCO-, -CONR-, -OCONR- , -NRNR-, -NRNRCO-, -NRCO-, -NRCO2-, -NRCONR-, -SO-, -SO2-, -NRSO2-, -SO2NR-, -NRSO2NR-, -O-, -S-; or -NR-; and every occurrence of R^W is independently R ', halogen, NO2, or CN, or -WR^Wis = O, = S, or = NR '.

ES 2 328 824 T3

In certain embodiments, Cy¹ is selected from one of the following rings:

<img file="ES2328824T3_D0019.tif" />

ES 2 328 824 T3

<img file="ES2328824T3_D0020.tif" />

It will be appreciated that, as generally described above, Cy¹ is optionally substituted on one or more carbon or nitrogen atoms with, and independent occurrences of, -Wr^w; y is 0-5; wherein W is a bond or is a C1-C6 alkylidene chain in which up to two methylene units of W are optionally and independently substituted with -CO-, -CO2-, -COCO-, -CONR-, -OCONR- , -NRNR-, -NRNRCO-, -NRCO-, -NRCO2-, -NRCONR-, -SO-, -SO2-, -NRSO2-, -SO2NR-, -NrSo2NR-, -O-, -S-; or -NR-; and every occurrence of R^W is independently R ', halogen, NO2, or CN, or -WR^W is = O, = S, or = NR '. In certain embodiments, y is 0, 1, 2, or 3, and W is a bond or is a C alkylidene chain₁ -C₆ optionally substituted wherein one or two methylene units are optionally and independently substituted with O, NR, S, SO₂, or CO₂, CO, and R^W is R 'or halogen. In other embodiments, y is 0, 1, 2, or 3

ES 2 328 824 T3 and every appearance of WR^W, when present, is independently -C 1-3 alkyl, -O (C1-3 alkyl), -CF3, -OCF3, -SCF3, -F, -Cl, -Br, -COOR ', -COR', -O (CH2) 2N (R) (R '), -O (CH2) N (R) (R'), -CON (R) (R '), - (CH2) 2OR', - (CH2) OR ', optionally substituted phenyl, optionally substituted benzyl, -N (R) (R '), - (CH₂)₂N (R) (R '), - (CH₂) n (R) (R ') -, or SO2NRR'.

In addition to the compounds and subsets described above, certain additional compounds are of interest.

For example, in certain embodiments, Z is CH₂, each R² and R³ is hydrogen, and R⁴ is V_pCy¹, where p is 1 and V is -CH2C (R⁶) 2 and the compounds have the general formula II:

<img file="ES2328824T3_D0021.tif" />

in which R¹, X, R⁶ and Cy¹ they are as generally defined above and in subsets herein.

As generally described above, X is S or O and compounds of formula II-A or II-B are provided:

<img file="ES2328824T3_D0022.tif" />

II-A II-B

In some embodiments, for compounds of general formulas II, II-A, or II-B, R¹ is selected from one of the following groups:

<td>^ - £ ^ - (QR^x) x</td><td>(QR^x) _L u (QR) x</td><td>N</td><td>H 3h^QRX&gt; N</td>

<td>to</td><td>b</td><td>C</td><td>d</td>

<td>(QR^x) x μΓ'Ίι *% -^N</td><td>(QR^x) x * V^N</td><td>n «'^qr,’«</td><td> ^/(°^RX) x</td>

<td>and</td><td>F</td><td>g</td><td>h</td>

<td>H. hrK i —- ü NJ x (R^xQ)</td><td>x (R^xQ) J—! I N H</td><td></td><td>(QR^X)_Xs /</td>

<td>i</td><td>j</td><td>k</td><td> 1</td>

ES 2 328 824 T3

<img file="ES2328824T3_D0023.tif" />

ES 2 328 824 T3

<img file="ES2328824T3_D0024.tif" />

In other embodiments R¹ is one of rings a, b, c, d, m, n, o, ee, gg, or pp. In other embodiments R¹ is ring a. In some other embodiments, R¹ is the gg ring.

In certain embodiments, for compounds of formulas II, II-A, or II-B, x is 0, 1, 2, or 3, and Q is a bond or is an optionally substituted C1-6 alkylidene chain in which one or two methylene units are optionally and independently substituted with O, NR, S, SO₂, or CO₂, CO, and RX is R 'or halogen. In other embodiments, x is 0, 1, 2, or 3 and each occurrence of QRX, when present, is independently -C alkyl_1-3, -O (C_1-3), -CF₃, -OCF₃, -SCF3, -F, -Cl, -Br, -COOR ', -COR', -O (CH₂) 2N (R) (R), -O (CH₂) N (R) (R '), -CON (R) (R'), - (CH ^ OR ', - &lt;CH₂) OR ', optionally substituted phenyl, optionally substituted benzyl, -N (R) (R'), - (CH₂)₂N (R) (R '), - (CH₂) N (R) (R ') -, or SO2NRR'.

In still other embodiments, for compounds of general formulas II, II-A, or II-B, R⁶ is hydrogen, methyl, or two occurrences of R⁶ on the same carbon atom they are taken together to form an optionally substituted 3-6 membered spiro ring having 0, 1 or 2 heteroatoms selected from nitrogen, oxygen, or sulfur.

In certain embodiments, for compounds of general formulas II, II-A, or II-B, Cy¹ is selected from one of the following rings:

<td>| -¡Í ^ r (WR^W)Y</td><td> ·&lt;</td><td>* ¿N</td><td>HJ¡- (WR^W)Y N</td>

<td>i</td><td>ii</td><td>iii</td><td>iv</td>

<td>(WR *% * V^N</td><td>(WR^w)_Y^s Yes-ⁿ</td><td>n «^{(wrW |}’</td><td>^ (WR^w)_Y</td>

<td>V</td><td>saw</td><td>Vil</td><td>viii</td>

ES 2 328 824 T3

<td>Η „Ν '^Ν. / R^wW)</td><td>and (R^WW) s ^NX,&gt; -! I N * H</td><td>JWV »^Nv</td><td>JWSA.</td>

<td>X</td><td>X</td><td>xi</td><td>xii</td>

<td>X &lt;~ j \ _ ~. (WR^W) and ν</td><td></td><td>* V</td><td>WR^wrS</td>

<td>xiii</td><td>xiv</td><td>XV</td><td>xvi</td>

<td>WR^wnX, Π Ν \ ^^S</td><td>WR^w&lt;A_N) = N xT</td><td>WR^w© N</td><td>WR^wAo J = N</td>

<td>xvii</td><td>xviii</td><td>xix</td><td>XX</td>

<td>WR^wor</td><td>0 6 \ -&gt; (WR^w)_Y</td><td>ιΌ ^^X(WR^w)_Y</td><td>| &lt;^ NH ** Ανη *%</td>

<td>xxi</td><td>xxii</td><td>xxiii</td><td>xxiv</td>

<td>H | - £ “~ (WR^w)_YH</td><td>&gt; χθχ | “Γ J- (WR^w)_YNH</td><td>K3^Xs* \ WR^w)_Y</td><td>V ^ - ^ ÍWR ^</td>

<td>XXV</td><td>xxvi</td><td>xxvii</td><td>xxviii</td>

<img file="ES2328824T3_D0025.tif" />

<img file="ES2328824T3_D0026.tif" />

ES 2 328 824 T3

<img file="ES2328824T3_D0027.tif" />

In certain embodiments, for compounds of formulas II, II-A, or II-B, y is 0-5; wherein W is a bond or is a C1-C6 alkylidene chain in which up to two methylene units of W are optionally and independently substituted with -CO-, -CO2-, -COCO-, -CONR-, -OCONR- , -NRNR-, -NRNRCO-, -NRCO-, -NRCO2-, -NRCONR-, -SO-, -SO₂-, -NRSO₂-, -SO₂NR-, -NRSO₂NR-, -O-, -S-; or -NR-; and every occurrence of R^W is independently R ', halogen, NO2, or CN, or -WR^W is = O, = S, or = NR '. In certain embodiments, y is 0, 1, 2, or 3, and W is a bond or is an optionally substituted C1-6 alkylidene chain in which one or two methylene units are optionally and independently substituted with O, NR, S, SO2, or CO2, CO, and R^W is R 'or halogen. In other embodiments, y is 0, 1, 2, or 3 and each occurrence of WR^W, when present, is independently -C 1-3 alkyl, -O (C_1-3), -OPh, -CF3, -OCF3, -SCF3, -F, -Cl, -Br, -COOR ', -COR', -O (CH₂&gt; ¡N (R) (R '), -O (CH₂) N (R) (R '), -CON (R) (R'), - (CH₂&gt; ¡OR ', - (CH₂) OR ', optionally substituted phenyl, optionally substituted benzyl, -N (R) (R'), - (CH₂)₂N (r) (r '), - (Ch₂) N (R) (R ') -, or SO2NRR'.

In still other embodiments, Z is a bond and R¹ is a saturated, partially unsaturated, or fully unsaturated 3-8 membered monocyclic ring having 0-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or a saturated, partially unsaturated, or 8-12 membered bicyclic ring system fully unsaturated having 0-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur; R² is hydrogen; R³ is UmR 'where m is 0 or 1, and U, when present, is -CH2- and R' is an optionally substituted saturated, partially unsaturated, or fully unsaturated 5 or 6-membered ring having 0-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur; and R⁴ is -CH2Cy¹ and compounds of formula III are provided:

<img file="ES2328824T3_D0028.tif" />

where R ', X, U, m, R', and Cy¹ they are as generally defined above and in subsets herein.

ES 2 328 824 T3

As generally described above, X is S or O and compounds of formula III-A or III-B are provided:

<img file="ES2328824T3_D0029.tif" />

In some embodiments, for compounds of general formulas III, III-A, or III-B, R¹ is selected from one of the following groups:

<img file="ES2328824T3_D0030.tif" />

ES 2 328 824 T3

<img file="ES2328824T3_D0031.tif" />

ES 2 328 824 T3

<img file="ES2328824T3_D0032.tif" />

In other embodiments R 'is one of rings a, b, c, d, m, n, o, ee, gg, or pp. In other embodiments R¹ is ring a. In some other embodiments, R¹ is the gg ring.

In certain embodiments, for compounds of formulas III, III-A, or III-B, x is 0, 1, 2, or 3, and Q is a bond or is a C alkylidene chain_1-6 optionally substituted wherein one or two methylene units are optionally and independently substituted with O, NR, S, SO₂, or CO₂, CO, and RX is R 'or halogen. In other embodiments, x is 0, 1, 2, or 3 and each occurrence of QRX, when present, is independently -C alkyl_1-3, -O (C_1-3), -CF₃, -OCF₃, -SCF₃, -F, -Cl, -Br, -COOR ', -COR', -O (CH₂) 2N (R) (R '), -O (CH₂) N (R) (R '), -CON (R) (R'), - (ch₂&gt; ¡Or ', - (CH₂) OR ', optionally substituted phenyl, optionally substituted benzyl, -N (R) (R'), - (CH₂)₂N (R) (R '), - (CH₂) N (R) (R ') -, or SO2NRR'.

As generally described above for compounds of formula III, R³ is U_mR 'where m is 0 or 1, and U, when present, is -CH₂- and R 'is an optionally substituted saturated, partially unsaturated, or fully unsaturated 5 or 6 membered ring having 0-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In certain embodiments, R 'is an optionally substituted phenyl or pyridyl group.

In certain embodiments, for compounds of general formulas III, III-A, or III-B, Cy¹ is selected from one of the following rings:

<td></td><td></td><td>and (R^WW) i li '^ N' n, β N</td><td>.N Η N</td>

<td> * 1</td><td>ii</td><td>iii</td><td>iv</td>

<td>(WR^w)_Y¹ V^N</td><td>(WR^W)Y i</td><td>.. (WR^w)_Y</td><td></td>

<td>V</td><td>saw</td><td>vii</td><td>viii</td>

<td>H í ^ΝΛ i_F(R^wW)</td><td>and (R^WW) H</td><td>_ff-|-5_r(WR^w),</td><td>aw »</td>

<td>ix</td><td>X</td><td>xi</td><td>xii</td>

ES 2 328 824 T3

<img file="ES2328824T3_D0033.tif" />

ES 2 328 824 T3

<img file="ES2328824T3_D0034.tif" />

In certain embodiments, for compounds of formulas III, III-A, or III-B, y is 0-5; where W is a bond or is a C alkylidene chain₁ -C₆ wherein up to two methylene units of W are optionally and independently substituted with -CO-, -CO₂- -COCO-, -CONR-, -OCONR-, -NRNR-, -NRNRCO-, -NRCO-, -nrco₂-, -nrconr-, -SO-, -SO₂-, -NRSO₂-, -SO₂NR-, -NRSO₂NR-, -O-, -S-; or -NR-; and every occurrence of R^W is independently R ', halogen, NO2, or CN, or -WR^W is = O, = S, or = NR '. In certain embodiments, y is 0, 1, 2, or 3, and W is a bond or is an optionally substituted Cj_6 alkylidene chain in which one or two methylene units are optionally and independently substituted with O, NR, S, SO₂, or CO₂, CO, and R^W is R 'or halogen. In other embodiments, y is 0, 1, 2, or 3 and each occurrence of WR^W, when present, is independently -C alkyl, ₃, -O (C_1-3), -CF₃, -OCF₃, -SCF₃, -F, -Cl, -Br, -COOR ', -COR', -O (CH₂) 2N (R) (R '), -O (CH₂) N (R) (R '), -CON (R) (R'), - (CH ^ OR ', - (CH2) OR', optionally substituted phenyl, optionally substituted benzyl, -N (R) (R ' ), - (CH₂)₂N (R) (R '), - (CH₂) N (R) (R ') -, or SO2NRR'.

In still other embodiments, ZR¹ is -CH3; R² is hydrogen; R³ is UmR 'where m is 0 or 1, and U, when present, is -CH₂- and R 'is an optionally substituted saturated, partially unsaturated, or fully unsaturated 5 or 6 membered ring having 0-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur; and R⁴ is -CH₂Cy¹ and compounds of formula IV are provided:

<img file="ES2328824T3_D0035.tif" />

where X, U, m, R ', and Cy¹ they are as generally defined above and in subsets herein.

As generally described above, X is S or O and compounds of formula IV-A or IV-B are provided:

<img file="ES2328824T3_D0036.tif" />

ES 2 328 824 T3

As generally described above for compounds of formula IV, R³ is UmR 'where m is 0 or 1, and U, when present, is -CH2- and R' is an optionally substituted saturated, partially unsaturated, or fully unsaturated 5 or 6-membered ring having 0-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In certain embodiments, R 'is an optionally substituted phenyl or pyridyl group.

In certain embodiments, for compounds of general formulas IV, IV-A, or IV-B, Cy¹ is selected from one of the following rings:

<td></td><td>'x.</td><td> ^^R«W ι-í V i N</td><td>^ - (WR), N</td>

<td>i</td><td>ii</td><td>iii</td><td>iv</td>

<td>(WR^w)_Yp ^ ii &lt;Qs n</td><td>(WR^w)_Yí (TA * \ -N</td><td> &gt;^NH</td><td>^ (WR^W)Y^HNJ?</td>

<td>V</td><td>saw</td><td>vii</td><td>viii</td>

<td>H í NN 1 W^Nand (R^WW)</td><td>/ R^wW) s ^N\, HL '^NH</td><td>.AAAA JtUWR '»),</td><td>ΛΑΛΑ. fl-K-jWR),^N'?</td>

<td>ix</td><td>X</td><td>xi</td><td>xii</td>

<td>A ^ - (WR^w)_Y</td><td>/ N^_t'x '(WR^w)_Y</td><td>| N - ^ (WR^w)_Yht_s&gt;</td><td>WR^wnA, II N \ ⁰</td>

<td>xiii</td><td>xiv</td><td>XV</td><td>xvi</td>

<td>WR^wor X</td><td>WR^w© N &lt;</td><td>WR^wS- ^ N</td><td>WR^wor S¿-N</td>

<td>xvii</td><td>xviii</td><td>xix</td><td>XX</td>

<td>WR^wÓ XT^N</td><td>/ Λ θ? V -&gt; (WR^w)_Y</td><td> '^^S^ (WR^w)Y</td><td>^t''^x- '(WR^w)_Y</td>

<td>xxi</td><td>xxii</td><td>xxiii</td><td>xxiv</td>

ES 2 328 824 T3

<img file="ES2328824T3_D0037.tif" />

In certain embodiments, for compounds of formulas IV, IV-A, or IV-B, y is 0-5; wherein W is a bond or is a C1-C6 alkylidene chain in which up to two methylene units of W are optionally and independently substituted with -CO-, -CO2-, -COCO-, -CONR-, -OCONR- , -NRNR-, -NRNRCO-, -NRCO-, -NRCO2, -NRCONR-, -SO-, -SO₂-, -NRSO₂-, -SO₂Nr-, -NrSo₂NR-, -O-, -S-; or -NR-; and every occurrence of R^W is independently R ', halogen, NO2, or CN, or -WR^W is = O, = S, or = NR '. In certain embodiments, y is 0, 1, 2, or 3, and W is a bond or is an optionally substituted C1-6 alkylidene chain one or two methylene units are optionally and independently substituted with O, NR, S, SO2, or CO2, CO, and R^W is R 'or halogen. In other embodiments, y is 0, 1, 2, or 3 and each occurrence of WR^W, when present, is independently -C alkyl_1-3, -O (C_1-3), -CF₃, -OCF₃, -SCF3, -F, -Cl, -Br, -COOR ', -COR', -O (CH2) 2N (R) (R '), -O (CH2) N (R) (R'), - CON (R) (R '), - (CH2) 2OR', - (CH2) OR ', optionally substituted phenyl, optionally substituted benzyl, -N (R) (R'), - (CH₂)₂N (R) (R '), - (CH₂) N (R) (R ') -, or SO2NRR'.

ES 2 328 824 T3

In still other embodiments, Z is -CH₂-; and R³ and R⁴, taken together with the nitrogen atom to which they are attached, form a saturated, partially unsaturated or fully unsaturated, optionally substituted 5, 6 or 7 membered ring, having the structure:

<img file="ES2328824T3_D0038.tif" />

<img file="ES2328824T3_D0039.tif" />

where r is 0, 1, or 2; one of X³, X⁴, or X⁵ is CH-Vp-Cy¹ or N-Vp-Cy¹, and the others of X³, X⁴, or X⁵ are CHR 'or NR'; every occurrence of X¹, when present, and X² is each independently C (R ')₂, -CO-, or-CS-, and compounds of formula V are provided:

<img file="ES2328824T3_D0040.tif" />

To i

V in which R¹, X, R², X¹, X², X³, X⁴, X⁵ and r are as generally described above and in subsets herein.

As generally described above, X is S or O and compounds of formula VA or

VB:

<img file="ES2328824T3_D0041.tif" />

In some embodiments, for compounds of general formulas V, VA, or VB, R¹ is selected from one of the following groups:

<td>H0- (qr^x) x</td><td>^ jj- (QR^X)«</td><td>μ N</td><td>.N H 3 ”(^qrX)&gt; N</td>

<td>to</td><td>b</td><td>c</td><td>d</td>

<td>(QR^X)« ⁵ V^N</td><td>(QR^X)_Xi Sf^N</td><td>n = x^(qrX)- 4^h</td><td>^ / (QR^x) x^h¥</td>

<td>and</td><td>F</td><td>g</td><td>h</td>

ES 2 328 824 T3

<img file="ES2328824T3_D0042.tif" />

ES 2 328 824 T3

<img file="ES2328824T3_D0043.tif" />

In other embodiments R¹ is one of rings a, b, c, d, m, n, o, ee, gg, or pp. In other embodiments R¹ is ring a. In some other embodiments, R¹ is the gg ring.

In certain embodiments, for compounds of formulas V, VA, or VB, x is 0, 1, 2, or 3, and Q is a bond or is an optionally substituted C1-6 alkylidene chain in which one or two methylene units they are optionally and independently substituted with O, NR, S, SO2, or CO2, CO, and RX is R 'or halogen. In other embodiments, x is 0, 1, 2, or 3 and each occurrence of QRX, when present, is independently -C 1-3 alkyl, -O (C1-3 alkyl), -CF3, -OCF3, -SCF3, - F, -Cl, -Br, -COOR ', -COR', -O (CH2) 2N (R) (R '), -O (CH2) N (R) (R'), -CON (R) ( R '), - (CH2) 2OR', - (CH2) OR ', optionally substituted phenyl, optionally substituted benzyl, -N (R) (R'), - (CH2) 2N (R) (R '), - (CH2) N (R) (R ') -, or SO2NRR'.

In certain embodiments, for compounds of general formulas V, VA, or VB, r is 1, each X¹, X², X³ and X⁵is CH2, and X⁴ is CH-Vp-Cy₁, or NV_p-Cy¹. In some other embodiments, p is 1 and V is SO₂, -NRSO₂, CO, or NRCO. In still other embodiments, X⁴ is N-Vp-Cy¹, p is 1 and V is SO2 or CO. In still other embodiments, for compounds of general formula VA or VB, X⁴ is N-Vp-Cy¹, p is 1 and V is SO2.

In certain embodiments, for compounds of general formulas V, VA, or VB, Cy¹ is selected from one of the following rings:

<img file="ES2328824T3_D0044.tif" />

ES 2 328 824 T3

<img file="ES2328824T3_D0045.tif" />

<img file="ES2328824T3_D0046.tif" />

<img file="ES2328824T3_D0047.tif" />

<img file="ES2328824T3_D0048.tif" />

<td>V</td><td>saw</td><td>vii</td><td>viii</td>

<td>Η , N'N. Η / y (R^WW)</td><td>/ R^wW) . ^N\. i_a N H</td><td>/ WV -jKwR'S V</td><td>ΛΑΛΑ * rl ^ -IWR), ^NV</td>

<td>ix</td><td>X</td><td>xi</td><td>xii</td>

<td>«'Q ^ (WR^w)_Y</td><td>/ N ^ (WR^w)_Y</td><td>i N ~ ax (^WRW)Y H_s&gt;</td><td>WR^wav^or</td>

<td>xiii</td><td>xiv</td><td>XV</td><td>xvi</td>

<td>WR^wn-4, &lt;^s'</td><td>WR^wor \ i) ®N</td><td>WR^wδ \ | Ά '</td><td>WR^wAA^N</td>

<td>xvii</td><td>xviii</td><td>xix</td><td>XX</td>

<td>WR^wAA^N</td><td>zy \ or 2 &gt; (WR^w)_Y</td><td>H © ^^X(WR^w)_Y</td><td>i í ^ nh HJ ^^z'(WR^w)_Y</td>

<td>xxi</td><td>xxii</td><td>xxiii</td><td>xxiv</td>

<td>H &lt;^ΝχΙ “Γ 4- (WR^w)_YN H</td><td>i pr 4- (WR^w)_YTO H</td><td>pro</td><td>..Λ Ya__r- (WR^w)_Y</td>

<td>XXV</td><td>xxvi</td><td>xxvii</td><td>xxviii</td>

ES 2 328 824 T3

<img file="ES2328824T3_D0049.tif" />

In certain embodiments, for compounds of general formulas V, VA, or VB, y is 0-5; wherein W is a bond or is a C1-C6 alkylidene chain in which up to two methylene units of W are optionally and independently substituted with -CO-, -CO2-, -COCO-, -CONR-, -OCONR- , -NRNR-, -NRNRCO-, -NRCO-, -NRCO2-, -NRCONR-, -SO-, -SO2-, -NRSO2-, -SO2NR-, -NRSO2NR-, -O-, -S-; or -NR-; and every occurrence of R^W is independently R ', halogen, NO2, or CN, or -WR^W is = O, = S, or = NR '. In certain embodiments, y is 0, 1, 2, or 3, and W is a bond or is an optionally substituted C1-6 alkylidene chain in which one or two methylene units are optionally and independently substituted with O, NR, S, SO2, or CO2, CO, and R^W is R 'or halogen. In other embodiments, y is 0, 1, 2, or 3 and each occurrence of WR^W, when present, is independently -C 1-3 alkyl, -O (C_1-3), -CF3, -OCF3, -SCF3, -F, -Cl, -Br, -COOR ', -COR', -O (CH₂) 2N (R) (R '), -O (CH₂) N (R) (R '), -CON (R) (R'), - (CH ^ OR ', - (CH₂) OR ', optionally substituted phenyl, optionally substituted benzyl, -N (R) (R'), - (CH₂)₂N (r) (r '), - (Ch₂) N (R) (R ') -, or SO2NRR'.

ES 2 328 824 T3

In another embodiment, the present invention provides compounds of formula VI:

<img file="ES2328824T3_D0050.tif" />

in which:

Ring A is a 3-7 membered ring having up to 3 heteroatoms selected from 0, S, or NR; R, R¹, Z, X, and Cy1 are as defined above.

As generally described above, X is S or O, and compounds of formula VI-A or VI-B are provided:

<img file="ES2328824T3_D0051.tif" />

In one embodiment, Ring A is a 3-7 membered cycloalkyl ring. In another embodiment, Ring A is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl. In certain embodiments, Ring A is cyclopentyl or cyclohexyl.

In another embodiment, Ring A has a ring oxygen heteroatom. Or, ring A has a sulfur heteroatom in the ring. Or ring A has a nitrogen heteroatom in the ring. Preferably R (in NR) is hydrogen.

In some embodiments, for compounds of general formulas VI, VI-A, or VI-B, R¹ is selected from one of the following groups:

<img file="ES2328824T3_D0052.tif" />

ES 2 328 824 T3

<img file="ES2328824T3_D0053.tif" />

ES 2 328 824 T3

<img file="ES2328824T3_D0054.tif" />

In other embodiments Rⁱ is one of rings a, b, c, d, m, n, o, ee, or pp. In other more Rⁱ is phenyl (ring a).

In certain embodiments, for compounds of formulas VI, VI-A, or VI-B, x is 0, 1, 2, or 3, and Q is a bond or is a C alkylidene chain_1-6 optionally substituted wherein one or two methylene units are optionally and independently substituted with O, NR, S, SO₂, or CO₂, CO, and RX is R 'or halogen. In other embodiments, x is 0, 1, 2, or 3 and each occurrence of QRX, when present, is independently -C alkyl_1-3, -O (C_1-3), -OPh, -CF₃, -OCF3, -SCF3, -F, -Cl, -Br, -COOR ', -COR', -O (CH₂) iN (R) (R '), -O (CH₂) N (R) (R '), -CON (R) (R'), - &lt;CH₂) íOR ​​', - (CH₂) OR ', optionally substituted phenyl, optionally substituted benzyl, -N (R) (R'), - (CH₂)₂N (r) (r '), - (Ch₂) N (R) (R ') -, or SO2NRR'.

In certain embodiments, for compounds of general formulas VI, VI-A, or VI-B, Cy¹ is selected from one of the following rings:

<img file="ES2328824T3_D0055.tif" />

ES 2 328 824 T3

<img file="ES2328824T3_D0056.tif" />

ES 2 328 824 T3

<img file="ES2328824T3_D0057.tif" />

<img file="ES2328824T3_D0058.tif" />

xxxvi xxxvií xxxvííi

<img file="ES2328824T3_D0059.tif" />

In one embodiment, Cy₁ is ring io ring xxxiii above.

In certain embodiments, for compounds of general formulas VI, VI-A, or VI-B, y is 0-5; where W is a bond or is a C alkylidene chain₁-C₆ wherein up to two methylene units of W are optionally and independently substituted with -CO-, -CO2-, -COCO-, -CONR-, -OCONR-, -NRNR-, -NRNRCO-, -NRCO-, -NRCO2- , -NRCONR-, -SO-, -SO2-, -NRSO2-, -SO2NR-, -NRSO2NR-, -O-, -S-; or -NR-; and every occurrence of R^W is independently R ', halogen, NO2, or CN, or -WR^W is = O, = S, or = NR '. In certain embodiments, y is 0, 1, 2, or 3, and W is a bond or is an optionally substituted C1-6 alkylidene chain in which one or two methylene units are optionally and independently substituted with O, NR, S, SO2, or CO2, CO, and R^W is R 'or halogen. In other embodiments, y is 0, 1, 2, or 3 and each occurrence of WR^W, when present, is independently -C alkyl_1-3, -O (C_1-3), -CF3, -OCF3, -SCF3, -F, -Cl, -Br, -COOR ', -COR', -O (CH₂) 2N (R) (R '), -O (CH₂) N (R) (R '), -CON (R) (R'), - (CH ^ OR ', - (CH₂) OR ', optionally substituted phenyl, optionally substituted benzyl, -N (R) (R'), - (CH₂)₂N (r) (r '), - (Ch₂) N (R) (R ') -, or SO2NRR'.

In one embodiment, Z is -CH2- or -CH2-CH2-. Or, Z is -CH2-.

In another embodiment, R¹ is phenyl optionally substituted with up to three substituents selected from -C alkyl_1-3, -O (C_1-3), -CF₃, -OCF₃, -SCF₃, -F, -Cl, -Br, -COOR ', -COR', -O (CH₂)₂N (R) (R '), -O (CH₂) N (R) (R '), -CON (R) (R'), - (CH₂)₂OR ', - (CH₂) OR ', optionally substituted phenyl, optionally substituted benzyl, -N (R) (R'), - (CH2) 2N (R) (R '), - (CH2) N (R) (R') -, or SO2NRR '.

Representative examples of the compounds of formula I are shown in Table 1 below.

ES 2 328 824 T3

Table 1. Examples of Compounds of Formula I:

<img file="ES2328824T3_D0060.tif" />

ES 2 328 824 T3 (continued)

<img file="ES2328824T3_D0061.tif" />

ES 2 328 824 T3 (continued)

<img file="ES2328824T3_D0062.tif" />

ES 2 328 824 T3 (continued)

<img file="ES2328824T3_D0063.tif" />

ES 2 328 824 T3 (continued)

<img file="ES2328824T3_D0064.tif" />

ES 2 328 824 T3 (continued)

<img file="ES2328824T3_D0065.tif" />

ES 2 328 824 T3 (continued)

<img file="ES2328824T3_D0066.tif" />

ES 2 328 824 T3 (continued)

<img file="ES2328824T3_D0067.tif" />

ES 2 328 824 T3 (continued)

<img file="ES2328824T3_D0068.tif" />

ES 2 328 824 T3 (continued)

<img file="ES2328824T3_D0069.tif" />

ES 2 328 824 T3 (continued)

<img file="ES2328824T3_D0070.tif" />

ES 2 328 824 T3 (continued)

<img file="ES2328824T3_D0071.tif" />

ES 2 328 824 T3 (continued)

<img file="ES2328824T3_D0072.tif" />

ES 2 328 824 T3 (continued)

<img file="ES2328824T3_D0073.tif" />

<img file="ES2328824T3_D0074.tif" />

ES 2 328 824 T3 (continued)

<img file="ES2328824T3_D0075.tif" />

ES 2 328 824 T3 (continued)

<img file="ES2328824T3_D0076.tif" />

<img file="ES2328824T3_D0077.tif" />

ES 2 328 824 T3 (continued)

<img file="ES2328824T3_D0078.tif" />

ES 2 328 824 T3 (continued)

<img file="ES2328824T3_D0079.tif" />

ES 2 328 824 T3 (continued)

<img file="ES2328824T3_D0080.tif" />

ES 2 328 824 T3 (continued)

<img file="ES2328824T3_D0081.tif" />

ES 2 328 824 T3 (continued)

<img file="ES2328824T3_D0082.tif" />

ES 2 328 824 T3 (continued)

<img file="ES2328824T3_D0083.tif" />

ES 2 328 824 T3 (continued)

<img file="ES2328824T3_D0084.tif" />

ES 2 328 824 T3 (continued)

<img file="ES2328824T3_D0085.tif" />

ES 2 328 824 T3 (continued)

<img file="ES2328824T3_D0086.tif" />

ES 2 328 824 T3 (continued)

<img file="ES2328824T3_D0087.tif" />

ES 2 328 824 T3 (continued)

<img file="ES2328824T3_D0088.tif" />

ES 2 328 824 T3

Four. General Synthetic Methodology

The compounds used in the present invention may generally be prepared by procedures known to those skilled in the art for analogous compounds, as illustrated by the general scheme below, and the preparative examples that follow.

Schemes I and II below represent the synthesis of thiazole and oxazole starting materials in which Z is -CH₂- and R¹ is optionally substituted phenyl.

Scheme I

<img file="ES2328824T3_D0089.tif" />

Scheme II

<img file="ES2328824T3_D0090.tif" />

References

Org. Lett., 2000, 2 (8), 1165-1168; AJ Phillips et al.

Chem Pharm Bull, 1986, 34 (7), 2851; K. Meguro et al.

ES 2 328 824 T3

Scheme III

Scheme III depicts general reaction conditions for the coupling of thiazole or oxazole starting materials (as generally represented above) and an appropriate amine to generate thiazole or oxazole amides as generally represented by the compounds of formula I.

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Although some exemplary embodiments have been represented and described hereinbefore and herein, it will be understood that the compounds of the invention may be prepared according to the procedures generally described above using the appropriate starting materials by procedures generally available to one of ordinary skill. in technique.

5. Uses, Formulation and Administration

Pharmaceutically acceptable compositions

As discussed above, the present invention provides compounds that are useful as modulators of ABC transporters and thus are useful in the treatment of diseases, disorders or conditions such as cystic fibrosis, hereditary emphysema, hereditary homochromatosis, deficiencies of coagulationcibrinolysis, such as protein C deficiency, Type 1 hereditary angioedema, lipid processing deficiencies, such as familial hypercholesterolemia, chylomicronemia Type 1, abetalipoproteinemia, lysosomal storage diseases, such as I cell disease / pseudo-Hurler, secretory diarrhea or polycystic kidney disease, mucopolysaccharidoses, Sandhof / Tay-Sachs, Crigler-Najjar type II, poliendocrinopathy / hyperinsulemia, diabetes mellitus, Laron's dwarfism, myeloperoxidase deficiency, primary hypoparathyroidism, melanoma, CDG type 1 glucanosis, hereditary emphysema, congenital hyperthyroidism, osteogenesis imperfecta, hereditary hypofibrinogenemia, ACT deficiency, Diabetes insipidus (DI), neurophyseal DI, neprogenic DI, Charcot-Marie Tooth syndrome, Perlizaeus-Merzbacher disease, neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, progressive supranuclear palsy, Pick's disease, various polyglutamine-related neurological disorders such as Huntington's spinocerebellar ataxia type I, spinal and bulbar muscular atrophy, dentatorubal pallidoluysian, and myotonic dystrophy, as well as spongiform encellophathies, such as hereditary Creutzfeldt-Jakob disease (due to a processing defect of prion proteins), Fabry disease, Straussler-Scheinker syndrome, COPD, dry eye disease, or Sjogren's disease.

Accordingly, in another aspect of the present invention, pharmaceutically acceptable compositions are used, wherein these compositions comprise any of the compounds described herein, and optionally comprise a pharmaceutically acceptable excipient, adjuvant, or vehicle. In certain embodiments, these compositions optionally further comprise one or more additional therapeutic agents.

It will also be appreciated that some of the compounds used in the present invention may exist in free form for treatment or, where appropriate, as a pharmaceutically acceptable derivative thereof. According to the present invention, a pharmaceutically acceptable derivative includes, but is not limited to, pharmaceutically acceptable salts, esters, salts of said esters, or any other adduct or derivative that after administration to a patient in need is capable of providing, directly or indirectly, a compound as otherwise described herein, or a metabolite or residue thereof.

As used herein, the term "pharmaceutically acceptable salt" refers to those salts which, within the scope of reasonable medical judgment, are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation. , allergic response and the like, and correspond to a reasonable benefit / risk ratio. A "pharmaceutically acceptable salt" refers to any non-toxic salt or salt of an ester of a compound of the present invention which, upon administration to a recipient, is capable of providing, directly or indirectly, a compound of the present invention or an active metabolite or remainder thereof.

Pharmaceutically acceptable salts are well known in the art. For example, SM Berge, et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, which is incorporated herein by reference. The pharmaceutically acceptable salts of the compounds herein

The invention includes those derived from suitable organic and inorganic acids and bases. Examples of non-toxic pharmaceutically acceptable acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, acid oxalic, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or using other procedures used in the art such as ion exchange. Other pharmaceutically acceptable salts include the adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphor sulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucerophosulfonate, glucerophosphate, glucerophosphate, fumarate, glycoheptophonate , heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, and undecanoate Similar. Salts derived from the appropriate bases include alkali metal, alkaline earth metal, ammonium, and N salts.⁺(C alkyl_1-4)₄. This invention also envisions the quaternization of any basic nitrogen-containing groups of the compounds described herein. Products soluble or dispersible in water or oil can be obtained by said quaternization. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Other pharmaceutically acceptable salts include, where appropriate, non-toxic ammonium, quaternary ammonium, and amine cations formed using counter ions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate, and aryl sulfonate.

As described above, the pharmaceutically acceptable compositions used in the present invention further comprise a pharmaceutically acceptable excipient, adjuvant, or carrier, which, as used herein, includes any and all solvents, diluents, or other. liquid vehicle, dispersion or suspension aids, surfactants, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as appropriate for the particular dosage form desired. Various excipients used in the formulation of pharmaceutically acceptable compositions and known techniques for the preparation thereof are described in Remington's Pharmaceutical Sciences, Sixteenth Edition, EW Martin (Mack Publishing Co., Easton, Pa., 1980). Except insofar as any conventional support medium is incompatible with the compounds of the invention, such as producing any undesirable biological effects or otherwise interacting in a detrimental manner with any other component or components of the pharmaceutically acceptable composition, their use is contemplated within the scope of the present invention. Some examples of materials that can serve as pharmaceutically acceptable excipients include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid. , or potassium sorbate, partial glyceride mixtures of saturated vegetable fatty oils, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, trisiliceous magnesium, polyvinyl pyrrolidone, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, lanolins, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; tragacanth powder; malt; jelly; talcum powder; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil; Sesame oil; olive oil; corn oil and soybean oil; glycols; such as a propylene glycol or polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid, pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffered solutions, as well as other compatible non-toxic lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweeteners, flavoring and perfuming agents, preservatives and antioxidants as well they may be present in the composition, according to the judgment of the formulator.

Uses of Pharmaceutically Acceptable Compounds and Compositions

A method of treating a condition, disease, or disorder involved in ABC transporter activity is described. Also described is a method for treating a condition, disease, or disorder involved in a deficiency of ABC transporter activity, the method comprising administering a composition comprising a compound of formula (I) to a subject, preferably a mammal, that does so. need.

Also described is a procedure to treat cystic fibrosis, hereditary emphysema, hereditary homochromatosis, coagulation-cyibrinolysis deficiencies, such as protein C deficiency, Type 1 hereditary angioedema, lipid processing deficiencies, such as familial hypercholesterolemia, Type 1 chylomicronemia, abetalipoproteinemia, lysosomal storage diseases, such as I cell / pseudo-Hurler disease, Secretory diarrhea or polycystic kidney disease, mucopolysaccharidoses, Sandhof / Tay-Sachs, Crigler-Najjar type II, poliendocrinopathy / hyperinsulemia, diabetes mellitus, Laron's dwarfism, myeloperoxidase deficiency, primary hypoparathyroidism, melanoma, CDG glucanosis type 1, hereditary emphysema, hyperthyroidism type 1 congenital, osteogenesis imperfecta, hereditary hypofibrinogenemia, ACT deficiency, Diabetes insipidus (DI), neurophyseal DI, neprogenic DI, Charcot-Marie Tooth syndrome, Perlizaeus-Merzbacher disease, neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, progressive supranuclear palsy, Pick's disease, various polyglutamine-related neurological disorders such as Huntington's spinocerebellar ataxia type I, spinal and bulbar muscular atrophy dentatorubal, pallidoluisian, and myo dystrophy

ES 2 328 824 tonic T3, as well as spongiform encellophathias, such as hereditary Creutzfeldt-Jakob disease (due to a prion protein processing defect), Fabry disease, Straussler-Scheinker syndrome, COPD, dry eye disease, or Sjogren's disease, comprising the step of administering to said mammal an effective amount of a composition comprising a compound of formula (I), or a preferred embodiment thereof as shown above.

Also described is a method for treating cystic fibrosis comprising the step of administering to said mammal a composition comprising the step of administering to said mammal an effective amount of a composition comprising a compound of formula (I), or a preferred embodiment of the same as shown above.

According to the invention an "effective amount" of the compound or pharmaceutically acceptable composition is that amount effective to treat or reduce the severity of one or more cystic fibrosis, hereditary emphysema, hereditary homochromatosis, coagulation-cyibrinolysis deficiencies, such as deficiency of protein C, hereditary angioedema Type 1, lipid processing deficiencies such as familial hypercholesterolemia, chylomicronemia Type 1, abetalipoproteinemia, Lysosomal storage diseases, such as I-cell disease / pseudo-Hurler, secretory diarrhea or polycystic kidney disease, mucopolysaccharidoses, Sandhof / Tay-Sachs, Crigler-Najjar type II, poliendocrinopathy / hyperinsulemia, Diabetes mellitus, Laron's dwarfism, myeloperoxidase deficiency, primary hypoparathyroidism, melanoma, CDG type 1 glucanosis, hereditary emphysema, congenital hyperthyroidism, osteogenesis imperfecta, hereditary hypofibrinogenemia, ACT deficiency, Diabetes insipidus (DI), neurophyseal DI, neprogenic DI, Charcot-Marie Tooth syndrome, Perlizaeus-Merzbacher disease, neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, progressive supranuclear palsy, Pick's disease, miscellaneous Polyglutamine-related neurological disorders such as Huntington's spinocerebellar ataxia type I, spinal and bulbar muscular atrophy, dentatorubal pallidoluysian, and myotonic dystrophy, as well as spongiform encelophaties, such as inherited Creutzfeldt-Jakob disease (due to a defect in prion protein processing), Fabry disease, Straussler-Scheinker syndrome, COPD, dry eye disease, or Sjogren.

The compounds and compositions, according to the described procedure can be administered using any amount and any effective route of administration to treat or reduce the severity of one or more of cystic fibrosis, hereditary emphysema, hereditary homochromatosis, coagulation-cyibrinolysis deficiencies, such as protein C deficiency, Type 1 hereditary angioedema, lipid processing deficiencies such as familial hypercholesterolemia, Type 1 chylomicronemia, abetalipoproteinemia, lysosomal storage diseases, such as I-cell / pseudo-Hurler disease, secretory diarrhea or polycystic kidney disease, mucopolysaccharidoses, Sandhof / Tay-Sachs, Crigler-Najjar type II, poliendocrinopathy / hyperinsulemia, diabetes mellitus, Laron's dwarfism, myeloperoxidase deficiency, primary hypoparathyroidism, melanoma, CDG glucanosis type 1, hereditary emphysema, congenital hyperthyroidism, osteogenesis imperfecta, Hereditary hypofibrinogenemia, ACT deficiency, Diabetes insipidus (DI), neurophyseal DI, neprogenic DI, Charcot-Marie Tooth syndrome, Perlizaeus-Merzbacher disease, neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, supranuclear palsy progressive, Pick's disease, various polyglutamine-related neurological disorders such as Huntington's spinocerebellar ataxia type I, spinal and bulbar muscular atrophy, dentatorubal pallidoluisian, and myotonic dystrophy, as well as spongiform encelophaties, such as inherited Creutzfeldt-Jakob disease (due to a prion protein processing defect), Fabry disease, Straussler-Scheinker syndrome, COPD , dry eye disease, or Sjogren's disease. The exact amount required will vary from subject to subject, depending on the species, age and general condition of the subject, the severity of the infection, the particular agent, its mode of administration, and the like. The compounds used in the invention are preferably formulated in unit dosage form for ease of administration and uniformity of a dosage. The term "unit dosage form" as used herein refers to a physically discrete unit of an agent appropriate for the patient to be treated. It will be understood, however, that the total daily use of the compounds and compositions of the present invention will be decided by the practicing physician within the scope of reasonable medical judgment. The specific effective dose level for any particular patient or organism will depend on various factors including the disorder to be treated and the severity of the disorder; the activity of the specific compound employed; the specific composition used; the age, body weight, general health, sex, and diet of the patient; the time of administration, the route of administration, and the rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincident with the specific compound employed, and similar factors well known in the medical arts. The term "patient", as used herein, refers to an animal, preferably a mammal, and more preferably a human.

The pharmaceutically acceptable compositions of the present invention can be administered to humans and other animals orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (such as in the form of powders, ointments, or drops), buccally, such as a oral or nasal spray, or the like, depending on the severity of the infection to be treated. In certain embodiments, the compounds of the invention can be administered orally or parenterally at dosage levels of from about 0.01 mg / kg to about 50 mg / kg and preferably from about 1 mg / kg to about 25 mg / kg, of body weight per day of the subject, one or more times per day, to obtain the desired therapeutic effect.

ES 2 328 824 T3

Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compounds, liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents, and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, acetate. ethyl, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, peanut, corn, germ, olive, castor and sesame), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and sorbitan fatty acid esters, and mixtures thereof. Besides inert diluents, oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Injectable preparations, for example sterile aqueous or oily injectable suspensions, can be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a parenterally acceptable non-toxic diluent or solvent, for example in the form of a 1,3-butanediol solution. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, USP, and isotonic sodium chloride solution. In addition, sterile, non-volatile oils are conventionally employed as a solvent or suspending medium. For this purpose, any soft non-volatile oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.

Injectable formulations can be sterilized, for example, by filtration through a bacteria retention filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use. .

To prolong the effect of a compound used in the present invention, it is often desirable to slow the absorption of the compound administered by subcutaneous or intramuscular injection. This can be achieved by using a liquid suspension of crystalline or amorphous material with poor solubility in water. The absorption rate of the compound therefore depends on its dissolution rate, which, in turn, may depend on the size of the crystal and the crystalline form. Alternatively, delayed absorption of a parenterally administered compound is achieved by dissolving or suspending the compound in an oil vehicle. Injectable depot forms are prepared by forming microencapsulated matrices of the compound in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of compound to polymer and the nature of the particular polymer employed, the release rate of the compound can be controlled. Examples of other biodegradable polymers include poly (orthoesters) and poly (anhydrides). Injectable depot formulations are also prepared by trapping the compound in liposomes or microemulsions that are compatible with body tissues.

Compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the compounds of the present invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax that are solid at room temperature but liquid at room temperature. from the body and therefore melt in the rectum or vaginal cavity and release the active compound.

Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In said solid dosage forms, the active compound is mixed with at least one inert, pharmaceutically acceptable excipient or vehicle, such as sodium citrate or dicalcium phosphate and / or) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, a) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as compounds quaternary ammonium, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbers such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents.

Solid compositions of a similar type can be employed as fillers in hard and soft filled gelatin capsules, using excipients such as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. Solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in pharmaceutical formulation engineering. They may optionally contain opacifying agents and may also be of a composition that they release the active ingredient (s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedded compositions that can be used include polymeric substances and waxes. Solid compositions of a similar type can also be used as fillers in hard and soft filled gelatin capsules, using excipients such as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.

ES 2 328 824 T3

The active compounds can also be in microencapsulated form with one or more excipients as indicated above. Solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, controlled release coatings, and other coatings well known in the pharmaceutical formulation art. In such solid dosage forms the active compound can be mixed with at least one inert diluent such as sucrose, lactose or starch. Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, for example tableting lubricants and other tableting aids such as magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. Optionally, they may contain opacifying agents and may also be of a composition that they release the active ingredient (s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedded compositions that can be used include polymeric substances and waxes.

Dosage forms for topical or transdermal administration of a compound of the present invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalers, or patches. The active component is mixed under sterile conditions with a pharmaceutically acceptable carrier and any required preservatives or buffers, as needed. Ophthalmic formulation, ear drops, and eye drops are also contemplated as within the scope of the present invention. Furthermore, the present invention contemplates the use of transdermal patches, which have the added advantage of providing controlled delivery of a compound to the body. Such dosage forms are prepared by dissolving or dosing the compound in the appropriate medium. Absorption enhancers can also be used to increase flux of the compound through the skin. The rate can be controlled by providing a rate control membrane or by dispersing the compound in a polymeric matrix or a gel.

As generally described above, the compounds used in the invention are useful as modulators of ABC transporters. Thus, without wishing to be bound by any particular theory, the compounds and compositions are particularly useful for treating or lessening the severity of a disease, condition, or disorder in which the hyperactivity or inactivity of ABC transporters is implicated in disease. condition, or disorder. When hyperactivity or inactivity of an ABC transporter is implicated in a particular disease, condition, or disorder, the disease, condition, or disorder may also be referred to as "ABC transporter-mediated disease, condition, or disorder." Accordingly, in another aspect, the present invention provides a method for treating or lessening the severity of a disease, condition, or disorder in which the hyperactivity or inactivity of an ABC transporter is implicated in said pathology.

The activity of a compound used in the present invention as a modulator of an ABC transporter can be assayed according to procedures generally described in the art and in the Examples herein.

It will also be appreciated that the pharmaceutically acceptable compounds and compositions used in the present invention can be employed in combination therapies, that is, the pharmaceutically acceptable compounds and compositions can be administered simultaneously with, before, or after one or more therapeutic compounds or medical procedures. desired. The particular combination of therapies (therapeutic compounds or procedures) to be employed in a combined regimen will take into account the compatibility of the desired therapeutic compounds and / or procedures and the desired therapeutic effect. It will also be appreciated that the therapies employed may achieve one desired effect for the same disorder (eg, a compound of the invention may be administered simultaneously with another agent used to treat the same disorder), or may achieve different effects (eg, control of any adverse effect). As used herein, additional therapeutic agents that are normally administered to treat or prevent a particular disease or condition are known to be "appropriate for the disease or condition to be treated."

The amount of additional therapeutic agent present in the compositions of the present invention will not be in an amount greater than that which would normally be administered in a composition comprising that therapeutic agent as the only active agent. Preferably, the amount of additional therapeutic agent in presently described compositions will range from about 50% to 100% of the amount normally present in a composition comprising that agent as the only therapeutically active agent.

The compounds used in the present invention or their pharmaceutically acceptable compositions can also be incorporated into compositions for coating an implantable medical device, such as prostheses, artificial valves, vascular grafts, stents and catheters. Accordingly, the present invention, in another aspect, includes an implantable device coating composition comprising a compound of the present invention as generally described above, and in the classes and subclasses herein, and a suitable vehicle for coating said implantable device. In yet another aspect, the present invention includes an implantable device coated with a composition comprising a compound of the present invention as generally described above, and in the classes and subclasses herein, and a suitable vehicle for the coating of said implantable device. Suitable coatings and general preparation of coated implantable devices are described in US Patents 6,099,562; 5,886,026; and 5,304,121. The liners are typically biocomposite polymeric materials.

ES 2 328 824 T3 ble such as a hydrogel type polymer, polymethyldisiloxane, polycaprolactone, polyethylene glycol, polylactic acid, ethylene vinyl acetate, and mixtures thereof. The coatings can optionally be further coated with a suitable topcoat of fluorosilicone, polysaccharides, polyethylene glycol, phospholipids, or combinations thereof to impart controlled release characteristics to the composition.

Another aspect of the invention refers to modulating the activity of the ABC transporter in a biological sample in vitro, a method that comprises contacting said biological sample with a compound of formula I or a composition comprising said compound. The term "biological sample" as used herein includes, without limitation, cell cultures or extracts thereof; biopsy material obtained from a mammal or extracts thereof; and blood, saliva, urine, feces, semen, tears, or other bodily fluids or extracts thereof.

Modulation of ABC transporter activity in a biological sample is useful for various purposes known to one of ordinary skill in the art. Examples of such purposes include, but are not limited to, the study of ABC transporters in biological and pathological phenomena; and the comparative evaluation of new modulators of ABC transporters.

In yet another embodiment, a method is provided for modulating the activity of an anion channel in vitro comprising the step of contacting said channel with a compound of formula (I). In preferred embodiments, the anion channel is a chloride channel or a bicarbonate channel. In other preferred embodiments, the anion channel is a chloride channel.

According to an alternative embodiment, the present invention provides a method for increasing the number of functional ABC transporters in a membrane of a cell in vitro, comprising the step of contacting said cell with a compound of formula (I). The term "functional ABC transporter" as used herein refers to an ABC transporter that is capable of transport activity. In preferred embodiments, said functional ABC transporter is CFTR.

According to another preferred embodiment, the activity of the ABC transporter is measured by measuring the potential of the transmembrane tension. The means for measuring the stress potential across a membrane in the biological sample may employ any of the procedures known in the art, such as an optical membrane potential assay or other electrophysiological procedures.

The optical membrane potential assay uses voltage sensitive FRET detectors described by Gonzalez and Tsien (See, Gonzalez, JE and RY Tsien (1995) "Voltage sensing by fluorescence resonance energy transfer in single cells" Biophys J 69 (4): 1272 -80, and Gonzalez, JE and RY Tsien (1997) "Improved indicators of cell membrane potential that use fluorescence resonance energy transfer" Chem Biol 4 (4): 269-77) together with instrumentation to measure changes in fluorescence such as the Voltage / Ion Probe Reader (VIPR ) (See, Gonzalez, JE, K. Oades, et al. (1999) "Cell-based assays and instrumentation for screening ion-channel targets" Drug Discov Today 4 (9): 431-439).

These stress-sensitive assays are based on the change in fluorescence resonant energy transfer (FRET) between the stress-sensitive, membrane soluble dye, DiSBAC.₂(3), and a fluorescent phospholipid, CC2-DMPE, which binds to the outer layer of the plasma membrane and acts as a FRET donor. Changes in membrane potential (V_m) cause the DiSBAC₂ (3) Negatively charged is redistributed across the plasma membrane and the amount of energy transfer from CC2-DMPE changes accordingly. Changes in fluorescence emission can be monitored using VIPR^TM II, which is an integrated fluorescence detector and liquid handler designed to perform cell-based scans in 96- or 384-well microtiter plates.

In order that the invention described herein may be more fully understood, the following examples are shown. It should be understood that these examples are for illustrative purposes only and are not intended to limit the present invention in any way.

Examples

A): Preparation of certain exemplary compounds used in the invention

General Experimental Procedures

Preparation of Amides: If the appropriate acid chloride was commercially available, it was added to one equivalent of the appropriate secondary amine in the minimum amount of 1,4-dioxane containing two equivalents of triethylamine. The reaction mixture was allowed to stir overnight at room temperature. The reaction mixture was then filtered and evaporated to dryness. The crude product was purified by reverse phase mass directed preparative liquid chromatography / mass spectrometry.

ES 2 328 824 T3

If the appropriate acid chloride was not commercially available, the appropriate carboxylic acid was added to a solution containing one equivalent of the appropriate amine in a minimal amount of acetonitrile containing two equivalents of triethylamine. O- (7-azabenzotriazol-1-yl) -N, N, N ', N'-tetramethyluronium hexafluorophosphate (HATU, 1.2 equiv.) Is added, and the reaction is stirred overnight. The crude product was then purified by reverse phase mass directed preparative liquid chromatography / mass spectrometry.

Specific Examples

General. All reagents and solvents were used as received without further purification. Thin layer chromatography was performed on 60 silica gel coated glass plates pre-coated with an EM Science fluorescent dye. Mass spectrometry was performed in the positive mode on a PE SCIEX EX150 mass spectrometer. Purity was determined by the observed total ion current and ultraviolet absorption at 220 nm and 254 nm.

Preparation of Amines

C- [1- (3,4-Dimethoxy-phenyl) -cyclopentyl] -methylamine: (3,4-dimethoxyphenyl) -acetonitrile (5.00 g, 28.2 mmol) was dissolved in 60 ml of anhydrous tetrahydrofuran in a 250 ml round bottom flask. Sodium hydride (2.03 g, 84.6 mmol) was added slowly and the reaction mixture was heated to 50-60 ° C. 1,4-Dichlorobutane (4.30 g, 33.9 mmol) was then added and the reaction mixture was refluxed for 16 hours. An additional aliquot of 1,4-dichlorobutane (4.30 g, 33.9 mmol) was added and the reaction mixture was refluxed for a further 24 hours. The reaction mixture was cooled to room temperature and quenched with the slow addition of methanol. The reaction mixture was evaporated to dryness and purified by silica gel column chromatography to yield a pale yellow oil (1.61 g, 6.98 mmol, 24.8%). The resulting 1- (3,4-dimethoxy-phenyl) -cyclopentanecarbonitrile (363 mg, 1.57 mmol) was dissolved in dry ether (4 ml) and cooled to 0 ° C under a nitrogen atmosphere. Lithium aluminum hydride (1.57 ml, 1M in ether) was added slowly and the reaction mixture was allowed to warm to room temperature and stir for 16 hours. The reaction mixture was quenched with the slow addition of methanol. The reaction mixture was washed with a saturated aqueous sodium chloride solution, separated and evaporated to dryness to give a colorless oil (356 mg, 1.38 mmol, 87.9%). ENI-MS m / z calc. 235.3, found 236.2 (M + 1)⁺. 1.64 minute retention time.

Preparation of Amides

2- (4-Methoxy-benzyl) -thiazole-4-carboxylic acid [1- (3,4-Dimethoxy-phenyl) -cyclopentylmethyl] -amide: 2- (4-methoxy-benzyl) -thiazole-4- carboxylic acid (101 mg, 0.405 mmol) and C- [1- (3,4-dimethoxy-phenyl) -cyclopentyl] -methylamine (96.5 mg, 0.410 mmol) were dissolved in acetonitrile (2 ml) containing triethylamine (84 , 1 pl, 0.600 mmol). O- (7-azabenzotriazol-1-yl) -N, N, N ', N'-tetramethyluronium hexafluorophosphate (171 mg, 0.450 mmol) was added and the solution was allowed to stir for 16 hours. The reaction mixture was evaporated to dryness and purified by silica gel column chromatography using a gradient of 5-30% ethyl acetate in hexanes. The pure fractions were combined and evaporated to dryness to give a yellow solid (84.0 mg, 0.180 mmol, 43.9%). ENI-MS m / z calc. 466.6, found 467.2 (M + 1)⁺. Retention time 8.32 minutes. NMR of¹H (400 MHz, CD₃CN) δ 1.68-2.07 (m, 8H), 3.50 (d, J = 6.3 Hz, 2H), 3.82 (s, 9H), 4.20 (s, 2H), 6.83-7.29 (m, 8H), 7.87 (s, 1H).

2-Benzyl-thiazole-4-carboxylic acid [2- (3,4-Dimethoxy-phenyl) -ethyl] -amide. Rink's amide type resin (0.627 g, 0.752 mmol, 1.20 mmol / g) was suspended and allowed to swell for 10 minutes in 4 ml of N, N-dimethylformamide (DMF). Phenylacetic acid (0.15 g, 1.1 mmol) and triethylamine (0.21 ml, 1.5 mmol) were then added to the reaction mixture. O- (7-azabenzotriazol-1-yl) -N, N, N ', N'-tetramethyluronium hexafluorophosphate (HATU, 0.46 g, 1.2 mmol) was added and the reaction mixture was stirred for two hours , filtered and washed with DMF and dichloromethane. The resin was then suspended in 5 ml of toluene and 2,4-bis- (4-methoxyphenyl) -1,3-dithia-2,4-diphosphetane-2,4-disulfide (Lawesson's reagent, 0.93 g, 2.3 mmol) to the suspension. The reaction mixture was then stirred for 2 hours at 65 ° C, filtered and washed with DMF and dichloromethane. The resin (0.50 g, 0.60 mmol) was then allowed to swell in 5 ml of tetrahydrofuran for 10 minutes and bromopyruvic acid (0.060 g, 0.36 mmol) was added to the reaction mixture. The mixture was then subjected to microwave irradiation for 20 minutes at 135 ° C, followed by filtration to yield crude 2-benzyl-thiazole-4-carboxylic acid. MS m / z calc. 219.0, found (ENI); 220.2 (M + H⁺). Retention time 2.38 minutes. The crude acid was dissolved in 2 ml of acetonitrile containing triethylamine (0.0836 ml, 0.600 mmol) and 2- (3,4-dimethoxy-phenyl) -ethylamine (0.0332 ml, 0.200 mmol). O- (7-azabenzotriazol-1-yl) -N, N, N ', N'-tetramethyluronium hexafluorophosphate (0.0836 g, 0.220 mmol) was added and the solution was allowed to stir for 16 hours. The reaction mixture was then purified by reverse phase preparative liquid chromatography to yield (0.011 g, 0.029 mmol, 4.8%) of a colorless oil. MS m / z calc. 382.1, found (ENI); 383.2 (M + H⁺) Retention time 2.97 minutes. NMR of¹H (400 MHz, CD₃CN) δ 2.85 (t, J = 7.0 Hz, 2 h), 3.53-3.65 (m, 2H), 3.78 (s, 3H), 3.80 (s, 3H), 4.33 (s, 2H), 6.78-6.91 (m, 3H), 7.29-7.42 (m, 5H), 7.52 (s, 1H ), 7.92 (s, 1H).

2- (4-Methoxy-benzyl) -oxazole-4-carboxylic acid [1- (3,4-Dimethoxy-phenyl) -cyclopentylmethyl] -amide

3-Hydroxy-2- [2- (4-methoxy-phenyl) -acetylamino] -propionic acid methyl ester: to a suspension of DL-serineHCl (2.0 g, 12.8 mmol) in dichloromethane (10 ml) at 0 ° C, triethylamine (3.58 ml, 25.7 mmol) and (4-methoxyphenyl) -acetyl chloride (1.96 ml, 12.8 mmol) were added dropwise. The mixture was stirred at 25 ° C for 12 hours. The mixture of

ES 2 328 824 T3 reaction was washed with a saturated aqueous solution of sodium chloride, separated and evaporated to dryness and purified by column chromatography (hexanes: ethyl acetate 25 to 100%). 3-Hydroxy2- [2- (4-methoxy-phenyl) -acetylamino] -propionic acid methyl ester was isolated as a white solid (2 g, 58%). ENI-MS m / z calc. 267.3, found 268.2 (M + 1)⁺. Retention time of 1.83 minutes. NMR of¹H (400 MHz, CDCl₃) 53.64 (s, 2H), 3.78 (s, 3H), 3.83 (s, 3H), 3.93 (cd, J = 14.6 and 4.0 Hz, 2H ), 4.66 (m, 1H), 6.40 (m, 1H), 7.01 (d, J = 8.7 Hz, 2H), 7.23 (d, J = 8.7 Hz, 2H).

2- (4-Methoxy-benzyl) -oxazole-4-carboxylic acid methyl ester: DAST (diethylamino sulfur trifluoride) was added to a cold (-78 ° C) solution of 3-hydroxy-2- [acid methyl ester]. 2- (4-methoxy-phenyl) -acetylamino] -propanoic acid (400 mg, 1.5 mmol) in dichloromethane (8 ml). After stirring for 1 h at -78 ° C, CCl₃Br (149 µl, 1.5 mmol) and DBU (226 µl, 1.5 mmol) and the reaction was allowed to warm to 25 ° C and stir at this temperature for 12 h. The reaction was poured into saturated aqueous NaHCO3 and the biphasic mixture was extracted with DCM. The combined organic extracts were dried over MgSO4 and purified by column chromatography (25 to 100% hexanes: ethyl acetate). 2- (4-Methoxybenzyl) -oxazole-4-carboxylic acid methyl ester was isolated as a white solid (80 mg, 21%). ENI-MS m / z calc. 247.5, found 248.2 (M + 1)⁺. Retention time of 2.64 minutes. NMR of¹H (400 MHz, CDCl3) δ 3.80 (s, 3H), 3.92 (s, 3H), 4.12 (s, 2H), 6.87 (d, J = 8.0 Hz , 2H), 7.24 (d, J = 8.0 Hz, 2H).

2- (4-Methoxy-benzyl) -oxazole-4-carboxylic acid: A mixture of 2- (4-methoxybenzyl) -oxazole-4-carboxylic acid methyl ester (40 mg, 0.16 mmol), methanol (2 ml) and 1N NaOH (1 ml) was heated to 60 ° C for 2 hours and allowed to stand at 25 ° C for 30 minutes. After dilution with water, the mixture was adjusted to pH 2 with 2N HCl and extracted with ether. The organic phases were combined, dried over MgSO₄ and evaporated to dryness to yield 25 mg (66%) of 2- (4-methoxy-benzyl) -oxazole-4-carboxylic acid as a white solid. ENI-MS m / z calc. 233.2, found 234.2 (M + 1)⁺. Retention time of 2.27 minutes.

2- (4-Methoxy-benzyl) -oxazole-4-carboxylic acid [1- (3,4-Dimethoxy-phenyl) -cyclopentylmethyl] -amide: starting from 2- (4-methoxy-benzyl) -oxazole- 4-carboxylic (25 g, 0.1 mmol) and [2- (3,4-dimethoxy-phenyl) -2-methyl] -propylamine (47 mg, 0.2 mmol) following a procedure similar to that presented for the preparation 2- (4-methoxy-benzyl) -thiazole-4-carboxylic acid [1- (3,4-dimethoxy-phenyl) -cyclopentylmethyl] -amine, the amide (30 mg, 66%) was obtained as a white solid. ENI-MS m / z calc. 450.5, found 451.2 (M + 1)⁺. Retention time of 3.51 minutes NMR of¹H (400 MHz, CDCl3) δ 1.68-2.19 (m, 8H), 3.50 (d, J = 6.3 Hz, 2H), 3.81 (s, 3H), 3 , 88 (s, 3H), 3.92 (s, 3H), 4.00 (s, 2H), 6.73-6.88 (m, 6H), 7.20 (d, J = 8.7 Hz, 2H), 8.07 (s, 1H).

Other compounds of formula I have been prepared by procedures substantially similar to those described above. Table 2 below shows analytical data for selected compounds of formula I.

TABLE 2

<td>No. of Comp.</td><td>LC-EM (M + 1)</td><td>CL-TR (min)</td><td></td><td>No. of Comp.</td><td>LC-EM (M + 1)</td><td>CL-TR (min)</td><td></td><td>No. of Comp.</td><td>LC-EM (M + 1)</td><td>CL-TR (min)</td>

<td> 6</td><td> 451,20</td><td> 3,60</td><td rowspan="7"></td><td> 21</td><td> 377,00</td><td> 4,46</td><td rowspan="7"></td><td> 32</td><td> 505,40</td><td> 4,24</td>

<td> 10</td><td> 468,00</td><td> 3,17</td><td> 22</td><td> 512,00</td><td> 4,57</td><td> 33</td><td> 489,20</td><td> 4,46</td>

<td> 12</td><td> 502,00</td><td> 3,25</td><td> 23</td><td> 501,00</td><td> 4,96</td><td> 34</td><td> 413,20</td><td> 3,62</td>

<td> 13</td><td> 473,00</td><td> 4,48</td><td> 24</td><td> 560,20</td><td> 4,33</td><td> 35</td><td> 427,00</td><td> 3,45</td>

<td> 14</td><td> 457,00</td><td> 4,66</td><td> 25</td><td> 323,00</td><td> 4,14</td><td> 36</td><td> 467,20</td><td> 4,23</td>

<td> 15</td><td> 454,00</td><td> 4,19</td><td> 26</td><td> 368,00</td><td> 4,12</td><td> 37</td><td> 451,20</td><td> 4,42</td>

<td> 16</td><td> 499,00</td><td> 4,25</td><td> 27</td><td> 354,00</td><td> 3,18</td><td> 38</td><td> 417,20</td><td> 3,87</td>

ES 2 328 824 T3

TABLE 2 (continued)

<td>No. of Comp.</td><td>LC-EM (M + 1)</td><td>CL-TR (min)</td><td></td><td>No. of Comp.</td><td>LC-EM (M + 1)</td><td>CL-TR (min)</td><td></td><td>No. of Comp.</td><td>LC-EM (M + 1)</td><td>CL-TR (min)</td>

<td> 17</td><td> 468,00</td><td> 3,17</td><td rowspan="4"></td><td> 28</td><td> 357,00</td><td> 3,90</td><td rowspan="4"></td><td> 39</td><td> 431,40</td><td> 3,73</td>

<td> 18</td><td> 468,00</td><td> 3,23</td><td> 29</td><td> 343,00</td><td> 3,40</td><td> 40</td><td> 471,20</td><td> 4,46</td>

<td> 19</td><td> 513,20</td><td> 3,16</td><td> 30</td><td> 479,00</td><td> 4,28</td><td> 41</td><td> 455,20</td><td> 4,68</td>

<td> 20</td><td> 422,00</td><td> 4,32</td><td> 31</td><td> 465,40</td><td> 3,52</td><td> 42</td><td> 437,20</td><td> 3,24</td>

<td> 43</td><td> 423,20</td><td> 3,40</td><td rowspan="20"></td><td> 63</td><td> 497,20</td><td> 3,58</td><td rowspan="20"></td><td> 83</td><td> 443,40</td><td> 3,51</td>

<td> 44</td><td> 361,00</td><td> 2,91</td><td> 64</td><td> 451,20</td><td> 3,77</td><td> 84</td><td> 481,40</td><td> 3,74</td>

<td> 45</td><td> 403,40</td><td> 3,25</td><td> 65</td><td> 451,40</td><td> 3,76</td><td> 85</td><td> 475,20</td><td> 4,09</td>

<td> 46</td><td> 417,20</td><td> 3,17</td><td> 66</td><td> 471,20</td><td> 3,72</td><td> 86</td><td> 392,00</td><td> 2,28</td>

<td> 47</td><td> 343,00</td><td> 3,53</td><td> 67</td><td> 505,40</td><td> 3,78</td><td> 87</td><td> 397,00</td><td> 3,60</td>

<td> 48</td><td> 457,40</td><td> 3,84</td><td> 68</td><td> 505,40</td><td> 3,80</td><td> 88</td><td> 391,00</td><td> 3,70</td>

<td> 49</td><td> 424,20</td><td> 2,53</td><td> 69</td><td> 505,20</td><td> 3,80</td><td> 89</td><td> 405,20</td><td> 3,84</td>

<td> 50</td><td> 423,20</td><td> 3,55</td><td> 70</td><td> 527,20</td><td> 3,35</td><td> 90</td><td> 421,00</td><td> 3,75</td>

<td> 51</td><td> 407,60</td><td> 3,71</td><td> 71</td><td> 481,40</td><td> 3,50</td><td> 91</td><td> 427,20</td><td> 3,85</td>

<td> 52</td><td> 437,40</td><td> 3,78</td><td> 72</td><td> 477,20</td><td> 3,48</td><td> 92</td><td> 405,20</td><td> 3,87</td>

<td> 53</td><td> 451,40</td><td> 3,64</td><td> 73</td><td> 526,00</td><td> 3,37</td><td> 93</td><td> 421,00</td><td> 3,73</td>

<td> 54</td><td> 418,20</td><td> 3,65</td><td> 74</td><td> 530,20</td><td> 3,62</td><td> 94</td><td> 425,00</td><td> 3,89</td>

<td> 55</td><td> 471,20</td><td> 3,67</td><td> 75</td><td> 439,40</td><td> 3,17</td><td> 95</td><td> 405,40</td><td> 3,87</td>

<td> 56</td><td> 501,20</td><td> 3,67</td><td> 76</td><td> 483,40</td><td> 2,90</td><td> 96</td><td> 421,20</td><td> 3,63</td>

<td> 57</td><td> 497,20</td><td> 3,49</td><td> 11</td><td> 441,20</td><td> 3,25</td><td> 97</td><td> 425,20</td><td> 3,89</td>

<td> 58</td><td> 437,20</td><td> 3,51</td><td> 78</td><td> 488,20</td><td> 3,03</td><td> 98</td><td> 451,00</td><td> 3,81</td>

<td> 59</td><td> 467,40</td><td> 3,63</td><td> 79</td><td> 492,20</td><td> 3,27</td><td> 99</td><td> 483,40</td><td> 4,12</td>

<td> 60</td><td> 467,40</td><td> 3,54</td><td> 80</td><td> 538,20</td><td> 3,32</td><td> 100</td><td> 481,20</td><td> 4,19</td>

<td> 61</td><td> 497,40</td><td> 3,33</td><td> 81</td><td> 451,20</td><td> 3,88</td><td colspan="3" rowspan="2"></td>

<td> 62</td><td> 497,40</td><td> 3,55</td><td> 82</td><td> 443,20</td><td> 3,51</td>

B) Assays to Detect and Measure AF508-RTFQ Correction Properties of Compounds

I) Optical Membrane Potential Procedures for Testing AF508RTFQ Modulating Properties of Compounds

The optical membrane potential assay used the voltage-sensitive FRET detectors described by Gonzalez and Tsien (See, Gonzalez, JE and RY Tsien (1995) “Voltage sensing by fluorescence resonance energy transfer in single cells” Biophys J 69 (4): 1272-80, and Gonzalez, JE and RY Tsien (1997) “Improved indicators of cell membrane potential that use fluorescence resonance energy transfer” Chem Biol 4 (4): 269-77) together with instrumentation to measure changes in fluorescence such as the Voltage / Ion Probe Reader (VIPR) (See, Gonzalez, JE, K. Oades, et al. (1999) "Cell-based assays and instrumentation for screening ion-channel targets" Drug Discov Today 4 (9): 431-439).

ES 2 328 824 T3

These stress-sensitive assays are based on the change in fluorescence resonant energy transfer (FRET) between the stress-sensitive, membrane soluble dye, DiSBAC.₂(3), and a fluorescent phospholipid, CC2-DMPE, which binds to the outer layer of the plasma membrane and acts as a FRET donor. Changes in membrane potential (V_m) cause the DiSBAC₂ (3) Negatively charged is redistributed across the plasma membrane and the amount of energy transfer from CC2-DMPE changes accordingly. Changes in fluorescence emission can be monitored using VIPR^TM II, which is an integrated fluorescence detector and liquid handler designed to perform cell-based scans in 96- or 384-well microtiter plates.

Identification of Corrective Compounds

To identify small molecules that correct the transit defect associated with AF508-CFTR, an HTS single addition assay format was developed. Cells were incubated in serum-free medium for 16 hours at 37 ° C in the presence or absence (negative control) of test compound. As a positive control, cells seeded in 384-well plates were incubated for 16 hours at 27 ° C until "temperature corrected" for AF508-CFTR. The cells were subsequently rinsed 3X with Krebs Ringers solution and loaded with the stress sensitive dyes. To activate AF508-CFTR, 10 pM forskolin and the CFRT enhancer, genistein (20 juM) were added, along with medium without Cl^- to each well. Adding medium without Cl^- promoted the discharge of Cl^- in response to the activation of AF508-CFTR and the resulting membrane depolarization was optically monitored using the FRET-based strain sensing dyes.

Identification of Enhancing Compounds

To identify enhancers of AF508-CFTR, an HTS double spike assay format was developed. During the first addition, a medium without Cl^-, with or without test compound, to each well. After 22 s, a second addition of medium without Cl was added.^- containing 2-10 pM forskolin to activate AF508-CFTR. The concentration of Cl^- extracellular after both additions was 28 mM, which promoted the discharge of Clin response to the activation of AF508-CFTR and the resulting membrane depolarization was optically monitored using the FRET-based strain sensing dyes.

<td></td><td>Solutions</td>

<td>Bath Solution N ° 1: (in mM) Bathroom Solution without Chloride:</td><td>NaCl 160, KCI 4.5, CaCI₂ 2, MgCI₂ 1, HEPES 10, pH 7.4 with NaOH. The chloride salts in Bath Solution # 1 are replaced by gluconate salts.</td>

<td>CC2-DMPE:</td><td>Prepared as a 10 mM stock solution in DEMO and stored at -20 ° C.</td>

<td>DiSBAC₂(3):</td><td>Prepared as a 10 mM stock solution in DEMO and stored at -20 ° C.</td>

Cell culture

NIH3T3 mouse fibroblasts stably expressing AF508-CFTR are used for optical measurements of membrane potential. Cells are kept at 37 ° C in a CO atmosphere₂ at 5% and 90% humidity in Dulbecco's modified Eagle's medium supplemented with 2 mM glutamine, 10% fetal calf serum, 1 X NEAA, / '- ME, 1 X pen / strep, and 25 mM HEPES in flasks of 175cm crop². For all optical assays, cells were seeded at 30,000 / well in matrigel-coated 384-well plates and cultured for 2 hours at 37 ° C before culturing at 27 ° C for 24 h to assay for enhancer. For correction assays, cells are grown at 27 ° C or 37 ° C with and without compounds for 16-24 hours.

B) Electrophysiological Assays to test the properties of modulation of AF508-CFTR of the compounds

1. Ussing Chamber Rehearsal

Experiments were performed in an Ussing chamber on polarized epithelial cells expressing AF508RTFQ to further characterize the AF508-CFTR modulators identified in the optical assays. Epithelial cells frt^af508-rtfq were grown in Costar Snapwell cell culture inserts mounted in an Ussing chamber (Physiologic Instruments, Inc., San Diego, CA), and the monolayers were continuously short-circuited using a tension clamp system (Department of Bioengineering, University of Iowa, IA , and, Physiologic Instruments, Inc., San Diego, CA). Transepithelial resistance was measured by applying a 2 mV pulse. Under these conditions,

ES 2 328 824 T3 epithelia of FRT demonstrated resistance of 4 ΚΩ / cm² or older. The solutions were kept at 27 ° C and bubbled with air. Electrode offset potential and fluid resistance were corrected using a cell-free insert. Under these conditions, the current reflects the flow of Cl^- through the AF508-CFTR expressed in the apical membrane. The I_SC was acquired digitally using an MP100A-CE interface and Acq Knowledge software (v3.2.6; BIOPAC Systems, Santa Barbara, CA).

Identification of Corrective Compounds

The typical protocol used a concentration gradient of Cl^- in membrane from basolateral to apical. To establish this gradient, normal Ringer's solution was used on the basolateral membrane, while the apical NaCl was replaced by equimolar sodium gluconate (titrated at pH 7.4 with NaOH) giving a large concentration gradient of Cl^- through the epithelium. All experiments were carried out with intact monolayers. To fully activate the AF508-CFTR, forskolin (10 µΜ) and the PDE inhibitor, IBMX (100 µΜ) were applied, followed by the addition of the CFTR enhancer, genistein (50 µM).

As seen in other cell types, low-temperature incubation of FRT cells stably expressing AF508-CFTR increases the functional density of CFTR at the plasma membrane. To determine the activity of the correction compounds, the cells were incubated with 10 µM of the test compound for 24 hours at 37 ° C and subsequently washed 3X before recording. The I_SC mediated by cAMP and genistein in cells treated with the compound was normalized to controls at 27 ° C and 37 ° C and expressed as a percentage of activity. Preincubation of cells with the correcting compound significantly increased cAMP- and genistein-mediated ISC compared to controls at 37 ° C.

Identification of Enhancing Compounds

The typical protocol used a concentration gradient of Cl^- in membrane from basolateral to apical. To establish this gradient, normal Ringer's solution was used on the basolateral membrane and was permeabilized with nystatin (360 μg / ml), while the apical NaCl was replaced by equimolar sodium gluconate (titrated at pH 7.4 with NaOH) giving a large concentration gradient of Cl^- through the epithelium. All experiments were performed 30 min after nystatin permeabilization. Forskolin (10 µM) and all test compounds were added to both sides of the cell culture inserts. The efficacy of the putative AF508-CFTR enhancers was compared with that of the known enhancer, genistein.

Solutions

Basolateral solution NaCl (135), CaCl2 (1,2), MgCl₂ (1,2), K₂HPO₄ (in mM): (2.4), KHPO₄ (0.6), N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) (10), and dextrose (10). The solution was titrated to pH 7.4 with NaOH.

Apical solution (in mM): Same as the basolateral solution, substituting NaCl for Na Gluconate (135).

Cell culture

Fisher rat epithelial cells (FRT) expressing AF508-CFTR (FRT^af508-rtfq) to perform Ussing chamber experiments for putative AF508-CFTR modulators identified from optical assays. Cells were grown in Costar Snapwell cell culture inserts and cultured for five days at 37 ° C and CO₂ 5% in Hams's F-12 modified by Coon medium supplemented with 5% fetal calf serum, 100 U / ml penicillin, and 100 µg / ml streptomycin. Before using it to characterize the enhancing activity of the compounds, cells were incubated at 27 ° C for 16-48 hours to correct for AF508-CFTR. To determine the activity of the correction compounds, the cells were incubated at 27 ° C or 37 ° C with and without the compounds for 24 hours.

2. Whole cell records

The macroscopic current of AF508-RTFQ (I_af508) in NIH3T3 cells with temperature corrected and test compound stably expressing AF508-CFTR were monitored using perforated patch whole cell recording. Briefly, tension clamp records of I_af508 at room temperature using an Axopatch 200B patch-clamp amplifier (Axon Instrumnts Inc., Foster City, CA). All records are

ES 2 328 824 T3 were acquired at a sampling frequency of 10 kHz and were low-pass filtered at 1 kHz. The pipettes had a resistance of 5-6 MQ when filled with the intracellular solution. Under these recording conditions, the inverse potential calculated for Cl^- (AND_C1) at room temperature was -28 mV. All registers had a seal resistance&gt; 20 GQ and a series resistance &lt;15 MQ. Pulse generation, data acquisition and analysis were performed using a PC equipped with a Digidata 1320 A / D interface in conjunction with Clampex 8 (Axon Instruments Inc.). The bath contained &lt;250 µl of saline and was continuously pre-fused at a rate of 2 ml / min using a gravity operated perfusion system.

Identification of Corrective Compounds

To determine the activity of the correction compounds to increase the density of functional AF508-CFTR in the plasma membrane, the perforated patch recording techniques described above were used to measure the current density after 24 h of treatment with the correction compounds. To fully activate AF508-CFTR, 10 pM of forskolin and 20 pM of genistein were added to the cells. Under these recording conditions, the current density after 24 h of incubation at 27 ° C was higher than that observed after 24 h of incubation at 37 ° C. These results are consistent with the known effects of low-temperature incubation at the density of AF508-CFTR on the plasma membrane. To determine the effects of correction compounds on CFTR current density, cells were incubated with 10 pM of the test compound for 24 hours at 37 ° C and the current density was compared with that of controls at 27 ° C. and 37 ° C (% activity). Before recording, cells were washed 3X with extracellular recording medium to remove any remaining test compound. Preincubation with 10 pM of correction compounds significantly increased the dependent current of cAMP and genistein compared to controls at 37 ° C.

Identification of Enhancing Compounds

The ability of AF508-RTFQ enhancers to increase Cl current was also investigated.^- AF508-RTFQ macroscopic (l_AF508) in NIH3T3 cells stably expressing AF508-CFTR using perforated patch recording techniques. Enhancers identified from optical testing caused a dose-dependent increase in l_AF508 with similar potency and efficacy seen in optical tests. In all cells examined, the reverse potential before and during the application of the enhancer was approximately -30 mV, which is the E_to calculated (-28 mV).

Solutions

Intracellular solution (in mM):

Extracellular solution (in mM):

Cs-aspartate (90), CsCI (50), MgCI₂ (1), HEPES (10), and 240 pg / ml amphotericin-B (pH adjusted to 7.35 with CsOH).

N-methyl-D-glucamine (NMDG) -CI (150), MgCI₂ (2), CaCI₂ (2), HEPES (10) (pH adjusted to 7.35 with HCl).

Cell culture

NIH3T3 mouse fibroblasts stably expressing AF508-CFTR were used for whole cell recordings. Cells are kept at 37 ° C in a CO atmosphere₂ at 5% and 90% humidity in Dulbecco's modified Eagle's medium supplemented with 2 mM glutamine, 10% fetal calf serum, 1 X NEAA, β-ME, 1 X pen / strep, and 25 mM HEPES in culture flasks 175 cm². For whole cell recordings, 2,5005,000 cells were seeded on poly-L-lysine coated glass coverslips and cultured for 24-48 hours at 27 ° C before use to assay for enhancers activity; and incubated with or without the correction compound at 37 ° C to measure the activity of the correctors.

3. Mono-channel registers

The mono-channel activities of temperature-corrected AF508-CFTR stably expressed in NIH3T3 cells and the activities of enhancer compounds were observed using an inside-out cleaved membrane patch. Briefly, single-channel activity strain-clamp recordings were made at room temperature with an Axopatch 200B patch-clamp amplifier (Axon Instruments Inc.). All recordings were acquired at a 10 kHz sample rate and low-pass filtered at 400 Hz. Patching pipettes were made from Corning Kovar Sealing No. 7052 glass (World Precision Instruments, Inc., Sarasota, FL ) and had a resistance of 5-8 MQ when filled with the extracellular solution. AF508-CFTR was activated after cleavage, adding 1 mM Mg-ATP, and 75 nM cAMP-dependent protein kinase, catalytic subunit (PKA; Promega Corp. Madison, Wl). After channel activity stabilized, the patch was perfused using a gravity-operated microperfusion system. The inflow became adjacent to the patch, resulting in

ES 2 328 824 T3 a complete solution exchange in 1-2 s. To maintain AF508-CFTR activity during rapid perfusion, the non-specific phosphatase F inhibitor (10 mM NaF) was added to the bath solution. Under these recording conditions, the channel activity remained constant for the duration of the patch recording (up to 60 min). The currents produced by positive charge moving from intracellular to extracellular solutions (anions moving in the opposite direction) are shown as positive currents. The potential of the pipette (V_p) was kept at 80 mV.

Channel activity was analyzed from membrane patches containing &lt;2 active channels. The maximum number of simultaneous openings determined the number of active channels during the course of an experiment. To determine the amplitude of single channel current, the recorded data from 120 s of AF508-RTFQ activity was filtered "off-line" at 100 Hz and then used to construct multi-point amplitude histograms that were fitted with multi-function functions. -gaussians using Bio-Patch Analysis software (Bio-Logic Comp. France). The total microscopic current and the open probability (P₀) were determined from 120 s of channel activity. The P₀ was determined using the Bio-Patch software or from the P ratio₀ = I / i (N), where I = average current, i = single channel current amplitude, and N = number of active channels in a patch.

Solutions

Extracellular solution (in mM):

Intracellular solution (in mM):

NMDG (150), Aspartic Acid (150), CaCl₂(5), MgCI₂ (2), and HEPES (10) (pH adjusted to 7.35 with Tris base).

NMDG-CI (150), MgCI₂ (2), EGTA (5), TES (10), and Tris base (14) (pH adjusted to 7.35 with HCl).

Cell culture

Mouse NIH3T3 fibroblasts stably expressing AF508-CFTR are used for membrane-cleaved patch-clamp recordings. Cells are kept at 37 ° C in a CO atmosphere₂ at 5% and 90% humidity in Dulbecco's modified Eagle's medium supplemented with 2 mM glutamine, 10% fetal calf serum, 1 X NEAA, β-ME, 1 X pen / strep, and 25 mM HEPES in culture flasks 175 cm². For mono-channel recordings, 2,500-5,000 cells were seeded on poly-L-lysine coated glass coverslips and cultured for 24-48 hours at 27 ° C prior to use.

The compounds of the invention are useful as modulators of ATP-binding cassette-type transporters, particularly CFTR. In certain embodiments, some of the compounds depicted in Table 1 exhibit a relative modulating efficacy of greater than 30%.