Solid forms of 3-(6-(1-(2,2-difluorobenzo[d][1,3] dioxol-5-yl) cyclopropanecarboxamido)-3-methylpyridin-2-yl) benzoic acid.

SOLID FORMS OF ACID 3- (6- (1- (2,2DIFLUOROBENZO [D] [1,3] DIOXOL-5-IL) CICLOPROPANCARBOXAMIDO) -3METILPIRIDIN-2 -ID) BENZOICO

FIELD OF THE INVENTION

The present invention relates to solid state forms, for example, crystalline forms of 3- (6- (1 (2,2-difluorobenzo [D] [1,3] dioxol-5-yl) cyclopropancarboxamido) acid -) 3-methylpyridin-2-yl) benzoic, pharmaceutical compositions thereof and methods therewith.

BACKGROUND OF THE INVENTION

CFTR is a cAMP / ATP mediated anion channel that is expressed in a variety of cell types, including absorptive and secretory epithelial cells, where it regulates the flow of anions through the membrane, as well as the activity of other ion channels and proteins. In epithelial cells, the normal function of CFTR is critical to maintaining the transport of electrolytes throughout the body, including the respiratory and digestive tissues. CFTR is composed of approximately 1,480 amino acids that encode a protein made from a tandem repeat of transmembrane domains, each containing six transmembrane helices and a nucleotide binding domain. The two transmembrane domains are linked by a large, polar, regulatory (R) domain with multiple phosphorylation sites that regulate channel activity and cell traffic.

The gene encoding 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 causes CFTR mutations resulting in cystic fibrosis (CF), the most common fatal genetic disease in humans. Cystic fibrosis affects about one in 2,500 children in the United States. Within the general population of the United States, up to 10 million people carry a single copy of the faulty gene with no apparent disease effects. In contrast, individuals with two copies of the CF-associated gene suffer from the impairment and fatal effects of CF, including chronic lung disease.

In cystic fibrosis patients, mutations in endogenously expressed CFTR in respiratory epithelia lead to reduced apical anion secretion causing an imbalance in ion and fluid transport. The resulting decrease in transport contributes to improved mucosal accumulation in the lung and accompanying microbial infections that ultimately cause death in CF patients. In addition to respiratory disease, CF patients typically suffer from gastrointestinal problems and pancreatic insufficiency that, if not treated, results in death. Also, most women with cystic fibrosis are infertile, and fertility is decreased among women with cystic fibrosis. Contrary to the serious effects of two copies of the CF-associated gene, individuals with a single copy of the CF-associated gene exhibit increased resistance to cholera and

<td>dehydration</td><td>which results from</td><td>the</td><td>diarrhea</td><td>- explaining</td>

<td colspan="2">maybe the frequency relatively</td><td>high</td><td>of the gene</td><td>CF inside</td>

<td>of the population. The analysis</td><td>of the sequence</td><td>of the</td><td>gene of</td><td>CFTR of</td>

CF chromosomes have revealed a variety of disease-causing 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. Nati. Acad. Sci. USA 87: 8447-8451). To date,&gt; 1000 disease-causing mutations in the CF gene have been identified (http: //www.genet.siclddds.onxa/cftr/). The most prevalent mutation is a phenylalanine deletion at position 508 of the CFTR amino acid sequence, and is commonly referred to as AF508-CFTR. This mutation occurs in approximately 70% of cystic fibrosis cases and is associated with severe disease.

Removal of residue 508 in AF508-CFTR prevents the nascent protein from folding properly. This results in the inability of the mutant protein to exit ER, and trafficking to the plasma membrane. As a result, the number of channels present in the membrane is much less than that observed in cells expressing wild-type CFTR. In addition to damaged traffic, the mutation results in control of the faulty channel. Together, the reduced number of channels in the membrane and poor control lead to reduced anion transport through the epithelia, leading to faulty fluid and ion transport. (Quinton, PM (1990), FASEB J. 4: 2709-2727). Studies have shown, however, that reduced numbers of AF508-CFTR in the membrane are functional, although lower than wild-type CFTR. (Dalemans et al. (1991), Nature Lond. 354: 526-528; Denning et al., Supra; Pasyk and Foskett (1995), J. Cell. Biochem. 270: 12347-50). In addition to ΔΡ508CFTR, other disease-causing mutations in CFTR that result in defective trafficking, synthesis, and / or channel control could be over- or under-regulated to alter anion secretion and modify disease progression and / or severity.

Although CFTR transports a variety of molecules to anions, it is clear that this role (the transport of anions) represents an element in an important mechanism for transporting ions and water through the epithelium. The other elements include the Na channel⁺ epithelial, ENaC, Na cotransporter⁺/ 2C1 '/ K⁺Na bomb⁺-K⁺- ATPasa and the K channels⁺ of the basolateral membrane, which are responsible for the uptake of chloride in the cell.

These elements work together to achieve directional transport through the epithelium through its expression and selective localization within the cell. Chloride absorption is carried out by the coordinated activity of ENaC and CFTR present in the apical membrane and the Na pump⁺-K⁺-ATPase and Cl channels expressed on the basolateral surface of the cell. The secondary active transport of chloride from the luminal side leads to the accumulation of intracellular chloride, which can then passively leave the cell through the Cl 'channels, resulting in vector transport. The Na co-transporter arrangement⁺/ 2C1 '/ K⁺Na bomb⁺-K⁺-ATPasa and K channels⁺ of the basolateral membrane on the basolateral surface and CFTR on the luminal side coordinate the secretion of chloride by means of CFTR on the luminal side. Because water is likely never to be actively transported by itself, its flow through epithelia depends on the small transepithelial osmotic gradients generated by the volumetric flow of sodium and chloride.

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

3- (6- (1- (2,2-Difluorobenzo [d] [1,3] dioxol-5yl) cyclopropancarboxamido) -3-methylpyridin-2-yl) benzoic acid in the salt form is described in PCT International WO 2007056341 (this publication being incorporated herein by reference in its entirety) as a modulator of CFTR activity and thus useful in the treatment of CFTR mediated diseases such as cystic fibrosis. However, there is a need for stable solid forms of such a compound that can be easily used in pharmaceutical compositions, suitable for use as therapeutics.

BRIEF DESCRIPTION OF THE INVENTION

The present invention relates to solid forms of 3- (6- (1- (2,2-difluorobenzo [d] [1,3] dioxol-5yl) cyclopropancarboxamido) -3-methylpyridin-2-yl) benzoic acid (in hereinafter Compound 1) having the following structure:

<img file="MX367154B_D0001.tif" />

Compound 1.

Compound 1 and the pharmaceutically acceptable compositions thereof are useful for treating or lessening the severity of cystic fibrosis. In one aspect, Compound 1 is in a substantially free and crystalline salt form referred to as Form 1 as described and characterized herein.

The processes described herein can be used to prepare the compositions of this invention comprising Form 1. The amounts and characteristics of the components used in the processes would be as described herein.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a calculated X-ray diffraction pattern of a single crystal structure of compound 1 in Form I.

Figure 2 is an actual X-ray diffraction pattern of Compound 1 in Form I.

Figure 3 is an overlay of a calculated X-ray diffraction pattern of a single crystal of Compound 1 in Form I, and an actual X-ray diffraction pattern of Compound 1 in Form I.

Figure 4 is a trace of differential scanning calorimetry (DSC) of compound 1 in Form I.

Figure 5 is a conformational image of Compound 1 in Form I based on single crystal X-ray analysis.

Figure 6 is a conformational image of Compound 1 in Form I based on X-ray analysis of single crystal as a dimer formed through carboxylic acid groups.

Figure 7 is a conformational image of Compound 1 in Form I based on single crystal X-ray analysis showing the molecules stacking together.

Figure 8 is a conformational image of Compound 1 in Form I based on single crystal X-ray analysis showing a different view (below a).

Figure 9 is an analysis of ^XH NMR of compound 1 in Form I in a suspension of 50 mg / mL, from methyl cellulose to 0.5-polysorbate 80 to T (O).

Figure 10 is an NMR analysis of Compound 1 in Form I in a 50 mg / mL suspension of methyl cellulose

<td>to the</td><td>0.5-polysorbate</td><td> 80</td><td>stored</td><td>to</td><td>temperature</td><td>environment</td>

<td colspan="2">for 24 hours. Figure 11 is</td><td>a</td><td>analysis of</td><td>^XH</td><td colspan="2">NMR of compound 1</td>

<td>HC1</td><td colspan="2">standard. DESCRIPTION</td><td>DETAILED OF</td><td>THE</td><td>INVENTION</td><td></td>

Definitions

As used herein, the following definitions will apply unless otherwise noted.

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

As used herein, crystalline refers to compounds or compositions where the structural units are arranged in fixed geometric patterns or networks, so that the crystalline solids have a long, rigid range order. The structural units that make up the crystal structure can be atoms, molecules or ions. Crystalline solids show definite melting points.

The term modular as used herein means increasing or decreasing, for example, activity, by a measurable amount.

In one aspect, the invention features a form of 3- (6- (1- (2,2-difluorobenzo [d] [1,3] dioxol-5-yl) cyclopropancarboxamido) -3-methylpyridin-2-yl) acid benzoic characterized by Form I.

In another embodiment, Form I is characterized by one or more peaks at 15.2 to 15.6 degrees, 16.1 to 16.5 degrees, and 14.3 to 14.7 degrees in an X-ray spray diffraction obtained using Cu Cu alpha radiation.

<td></td><td>In</td><td>other</td><td>modality, the Form</td><td>I</td><td>I know</td><td>characterizes</td><td colspan="2">for one or</td>

<td>plus</td><td colspan="2">! spikes in In other</td><td colspan="3">15.4, 16.3 and 14.5 degrees, modality, Form I is</td><td>characterizes</td><td>also</td><td>by</td>

<td>a</td><td>peak In</td><td>of 14 other</td><td>.6 to 15.0 degrees. modality, the Form</td><td>I</td><td>I know</td><td>characterizes</td><td>also</td><td>by</td>

<td>a</td><td>peak In</td><td>to 14. other</td><td>8 degrees. modality, the Form</td><td>I</td><td>I know</td><td>characterizes</td><td>also</td><td>by</td>

<td>a</td><td>peak</td><td>at 17.6 to 18.0 degrees.</td><td></td><td></td><td></td><td></td><td></td>

<td></td><td>In</td><td>another modality, the Form</td><td>I</td><td>I know</td><td>characterizes</td><td>also</td><td>by</td>

<td>a</td><td>peak</td><td>at 17.8 degrees.</td><td></td><td></td><td></td><td></td><td></td>

<td></td><td>In</td><td>another modality, the Form</td><td>I</td><td>I know</td><td>characterizes</td><td>also</td><td>by</td>

<td>a</td><td>peak</td><td>from 16.4 to 16.8 degrees.</td><td></td><td></td><td></td><td></td><td></td>

<td></td><td>In</td><td>another modality, the Form</td><td>I</td><td>I know</td><td>characterizes</td><td>also</td><td>by</td>

<td>a</td><td>peak</td><td>from 16.4 to 16.8 degrees.</td><td></td><td></td><td></td><td></td><td></td>

<td></td><td>In</td><td>another modality, the Form</td><td>I</td><td>I know</td><td>characterizes</td><td>also</td><td>by</td>

<td>a</td><td>peak</td><td>at 16.6 degrees.</td><td></td><td></td><td></td><td></td><td></td>

<td></td><td>In</td><td>another modality, the Form</td><td>I</td><td>I know</td><td>characterizes</td><td>also</td><td>by</td>

<td>a</td><td>peak</td><td>from 7.6 to 8.0 degrees.</td><td></td><td></td><td></td><td></td><td></td>

<td></td><td>In</td><td>another modality, the Form</td><td>I</td><td>I know</td><td>characterizes</td><td>also</td><td>by</td>

<td>a</td><td>peak</td><td>at 7.8 degrees.</td><td></td><td></td><td></td><td></td><td></td>

<td></td><td>In</td><td>another modality, the Form</td><td>I</td><td>I know</td><td>characterizes</td><td>also</td><td>by</td>

<td>a</td><td>peak</td><td>from 25.8 to 26.2 degrees.</td><td></td><td></td><td></td><td></td><td></td>

<td></td><td>In</td><td>another modality, the Form</td><td>I</td><td>I know</td><td>characterizes</td><td>also</td><td>by</td>

<td>a</td><td>peak</td><td>at 26.0 degrees.</td><td></td><td></td><td></td><td></td><td></td>

<td></td><td>In</td><td>another modality, the Form</td><td>I</td><td>I know</td><td>characterizes</td><td>also</td><td>by</td>

<td>a</td><td>peak</td><td>from 21.4 to 21.8 degrees.</td><td></td><td></td><td></td><td></td><td></td>

<td></td><td>In</td><td>another modality, the Form</td><td>I</td><td>I know</td><td>characterizes</td><td>also</td><td>by</td>

<td>a</td><td>peak</td><td>at 21.6 degrees.</td><td></td><td></td><td></td><td></td><td></td>

<td></td><td>In</td><td>another modality, the Form</td><td>I</td><td>I know</td><td>characterizes</td><td>also</td><td>by</td>

<td>a</td><td>peak</td><td>from 23.1 to 23.5 degrees.</td><td></td><td></td><td></td><td></td><td></td>

<td></td><td>In</td><td>another modality, the Form</td><td>I</td><td>I know</td><td>characterizes</td><td>also</td><td>by</td>

a peak at 23.3 degrees.

In some embodiments, Form I is characterized by a diffraction pattern substantially similar to that in Figure

1.

In some embodiments, Form I is characterized by a diffraction pattern substantially similar to that of Figure 2.

In some embodiments, the D90 particle size distribution is approximately 82 pm or less for Form I.

In some embodiments, the D50 particle size distribution is approximately 30 pm or less for Form I.

In one aspect, the invention features a pharmaceutical composition comprising Form I and a pharmaceutically acceptable carrier.

In one aspect, the present invention features a method for treating CFTR-mediated disease in a human, which comprises administering to the human an effective amount of Form I.

In some embodiments, the method comprises administering an additional therapeutic agent.

In some modalities, the disease is selected from cystic fibrosis, hereditary emphysema, hereditary hemochromatosis, coagulation-fibrinolysis deficiencies, such as protein C deficiency, hereditary type 1 angioedema, lipid processing deficiencies, such as familial hypercholesterolemia, chylomicronemia of type 1, abetalipoproteinemia, lysosomal storage diseases, such as l / pseudo-Hurler cell disease, mucopolysaccharidosis, Sandhof / Tay-Sachs, Crigler-Najjar type II, poliendocrinopatia / hiperinsulemia, diabetes mellitus, Laron dwarfism, mileoperoxidase deficiency, primary hypoparathyroidism, melanoma, hereditary emphysema, congenital hyperthyroidism, hypophyrine, hypophyrine, hyperthyroidism ACT deficiency, diabetes insipidus (DI), neurofiseal DI, neprogenic DI, Charcot-Marie dental syndrome, Perlizaeus-Merzbacher disease, neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, progressive supranuclear plasia, Pick disease, various polyglutamine neurological disorders such as Huntington, type I spinocerebular ataxia, spinal and bulbar muscular atrophy, dentatorubal palidoluisian, and myotonic dystrophy as well such as spongiform encephalopathies, such as hereditary Creutzfeldt-Jakob disease, Fabry disease, Straussler-Scheinker syndrome, COPD, dry eye syndrome and Sjogren's disease.

In one embodiment, the present invention provides a method for treating cystic fibrosis in a human, which comprises administering to such a human an effective amount of Form I.

In one aspect, the present invention features a kit comprising Form I and instructions for use thereof.

In one aspect, the present invention features a process for preparing Form I which comprises dispersing or dissolving 3- (6- (1- (2,2difluorobenzo [d] [1,3] dioxol-5-) HC1 salt yl) cyclopropancarboxamido) -3methylpyridin-2-yl) benzoic in an appropriate solvent for an effective amount of time.

In one embodiment, the present invention features a process for preparing Form I which comprises dispersing the HC1 salt of 3- (6- (1- (2,2difluorobenzo [d] [1,3] dioxol-5-yl) acid cyclopropancarboxamido) -3methylpyridin-2-yl) benzoic in an appropriate solvent for an effective amount of time.

In some embodiments, the appropriate solvent is water or an alcohol / water mixture.

In some embodiments, the appropriate solvent is water or a 50% methanol / water mixture.

In some embodiments, the appropriate solvent is water.

In some embodiments, the appropriate solvent is a mixture comprising 50% methanol and 50% water.

In some modalities, the effective amount of time is from about 2 to about a day. In some embodiments, the effective amount of time is from about 2 to about hours. In some embodiments, the effective amount of time is from about 2 to about hours. In some embodiments, the effective amount of me is about 2 to about 6 hours.

In one aspect, the invention features a crystalline form of the acid

3- (6- (1- (2,2difluorobenzo [d] [1,3] dioxol-5-yl) cyclopropancarboxamido) -3methylpyridin-2-yl) benzoic having a crystal to monoclinic system, a space group P2i / n and the following unit cell dimensions: a = 4.9626 (7) Á, b = 12.2994 (18) A, c = 33.075 (4) Á, 0 (= 90 °, β = 93.938 (9) ° and Y = 90 ° .

Methods for Preparing Form I.

In one embodiment, Form I is prepared from the dispersion or solution of a salt form, such as HC1, of the 3- (6- (1- (2,2-difluorobenzo [d] [1,3] dioxol) acid -5yl) cyclopropancarboxamido) -3-methylpyridin-2-yl) benzoic in an appropriate solvent for an effective amount of time. In another embodiment, Form I is prepared from the dispersion of the salt form, such as HC1, of the 3- (6 (1- (2,2-difluorobenzo [d] [1,3] dioxol-5yl) acid) cyclopropancarboxamido) -3-methylpyridin-2-yl) benzoic in an appropriate solvent for an effective amount of time. In another embodiment, Form I is formed directly from 3- (6- (1- (2,2-difluorobenzo [d] [1,3] dioxol-5yl) cyclopropancarboxamido) -3-methylpyridin-2- benzoate. yl) -t-butyl and an appropriate acid, such as formic acid. In one embodiment, the form of the HC1 salt of 3- (6- (1- (2,2difluorobenzo [d] [1,3] dioxol-5-yl) cyclopropancarboxamido) -3methylpyridin-2-yl) benzoic acid is the starting point and in one embodiment can be prepared by coupling an acid chloride radical with an amine according to Reaction Schemes 1-3.

Reaction Scheme 1. Synthesis of the chloride radical

one. Acid reduction.

<img file="MX367154B_D0002.tif" />

------------&gt;·

2. NaOH

<img file="MX367154B_D0003.tif" />

one. SOC1₂

<img file="MX367154B_D0004.tif" />

<img file="MX367154B_D0005.tif" />

<img file="MX367154B_D0006.tif" />

KOH

one. NaCN *

2, H_;OR

<img file="MX367154B_D0007.tif" />

SOClj

Ί

<img file="MX367154B_D0008.tif" />

Reaction Scheme 2

Synthesis of the amine radical

<img file="MX367154B_D0009.tif" />

N Br (Hoya

<img file="MX367154B_D0010.tif" />

C0₂®m

one. K₂CO₃, Pd (dppf) Cl_:

2. MsOH ac.

3. NaOH ac.

<img file="MX367154B_D0011.tif" />

OOjtBu urea-hydrogen peroxide phthalic anhydride

EtOAc, water

HjN

<img file="MX367154B_D0012.tif" />

CO ^ íBu

one. More₂O, py, MeCN

2. ethanolamine

<img file="MX367154B_D0013.tif" />

COjtBw

Reaction Scheme 3. Formation of an acidic salt of 3- (6- (1- (2,2-difluorobenzo [d] [1,3] dioxol-5yl) cyclopropancarboxamido) -3-methylpyridin-2-yl) benzoic acid .

<img file="MX367154B_D0014.tif" />

TFA, cat DMAP

Ν '

CO₂®u

Acidic fo

COjtBu

FO acid CO ^ H

Using HCI, for example, the salt form of 3 (6- (1- (2,2-difluorobenzo [d] [1,3] dioxol-5yl) cyclopropancarboxamido) -3-methylpyridin-2-yl) benzoic acid as Starting point, Form I can be formed in high yields by dispersing or dissolving the HC1 salt form of 3- (6- (1- (2,2-difluorobenzo [d] [1,3] dioxol-5yl) cyclopropancarboxamido acid ) -3-methylpyridin-2-yl) benzoic in an appropriate solvent for an effective amount of time. Other salt forms of 3- (6- (1- (2,2-difluorobenzo [d] [1,3] dioxol-5-yl) cyclopropancarboxamido) -3-methylpyridin-2-yl) benzoic acid, such as, for example, can be used other forms of mineral or organic acids. The other salt forms result from the hydrolysis of t-butyl ester with the corresponding acid. Other forms of acids / salts include nitric, sulfuric, phosphoric, boric, acetic, benzoic, malonic acid and the like. The salt form of 3 (6- (1- (2,2-difluorobenzo [d] [1,3] dioxol-5yl) cyclopropancarboxamido) -3-methylpyridin-2-yl) benzoic acid may or may not be soluble depending on the solvent used, but the lack of solubility does not prevent the formation of Form I. For example, in one embodiment, the appropriate solvent may be water or an alcohol / water mixture, such as a 50% methanol / water mixture, although the HC1 salt form of the 3- (6- (1 - (2,2-Difluorobenzo [d] [1,3] dioxol-5yl) cyclopropancarboxamido) -3-methylpyridin-2-yl) benzoic is only slightly soluble in water. In one embodiment, the appropriate solvent is water.

The effective amount of time for the formation of Form I of the 3- (6- (1- (2,2 difluorobenzo [d] [1,3] dioxol-5-yl) cyclopropancarboxamido) -3-methylpyridine- 2-yl) benzoic can be any time between 2 to 24 hours or more. In general, more than 24 hours is not necessary to obtain high yields (-98%), but some solvents may require longer amounts of time. It is also recognized that the amount of time required is inversely proportional to temperature. That is, the higher

<td>temperature</td><td>minor the</td><td>time required</td><td>to effect</td><td>the</td>

<td>dissociation</td><td>of acid</td><td>to form the</td><td>Form I. When</td><td>the</td>

<td>solvent is</td><td>water, the</td><td>agitation of the</td><td colspan="2">dispersion during</td>

approximately 24 hours at room temperature gives Form I in a yield of approximately 98%. If a solution of the saline form of 3- (6- (1- (2,2difluorobenzo [d] [1,3] dioxol-5-yl) cyclopropancarboxamido) -3methylpyridin-2-yl) benzoic acid is desired for the purposes of the process, a high temperature can be used. After stirring the solution for an effective amount of time at elevated temperature, cooling recrystallization produces the substantially pure forms of Form I. In one embodiment, substantially pure refers to more than about 90% purity. In another embodiment, "substantially pure" refers to more than about 95% purity. In another embodiment, "substantially pure" refers to more than about 98% purity. In another embodiment, "substantially pure" refers to more than about 99% purity. The selected temperature depends in part on the solvent used and is well within the capabilities of one skilled in the art of determining. In one embodiment, the temperature is between room temperature and approximately 80 ° C. In another embodiment, the temperature is between room temperature and approximately 40 ° C. In another embodiment, the temperature is between about 40 ° C and about 60 ° C. In another embodiment, the temperature is between about 60 ° C and about 80 ° C.

In some embodiments, Form I can be further purified by recrystallization from an organic solvent. Examples of organic solvents include, but are not limited to, toluene, eumene, anisole, 1-butanol, isopropyl acetate, butyl acetate, isobutyl acetate, methyl t-butyl ether, methyl isobutyl ketone, or 1-propanol / water (to different relationships). The temperature can be used as previously described. For example, in one embodiment, Form I is dissolved in 1-butanol at 75 ° C until completely dissolved. Cooling the solution to 10 ° C at a rate of 0.2 ° C / min produces Form I crystals that can be isolated by filtration.

Uses, formulation and administration

Pharmaceutically Acceptable Compositions

In another aspect of the present invention, pharmaceutically acceptable compositions are provided, wherein these compositions comprise Form I as described herein, and optionally comprise a pharmaceutically acceptable carrier, adjuvant, or vehicle. In some embodiments, these compositions optionally further comprise one or more additional therapeutic agents.

As described above, the pharmaceutically acceptable compositions of the present invention further comprise a pharmaceutically acceptable carrier, adjuvant, or vehicle, which, as used herein, includes any and all of solvents, diluents, or other liquid carriers, dispersion aids, or suspension, surfactants, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as appropriate to the particular dosage form desired. Remington's Pharmaceutical Sciences, Sixteenth Edition, EW Martin (Mack Publishing Co., Easton, Pa., 1980) describes different vehicles used in the formulation of the pharmaceutically acceptable compositions and the known techniques for their preparation. Except insofar as any conventional vehicle medium is incompatible with the compounds of the invention, such as producing any undesirable biological effect or otherwise, interacting in a detrimental manner with any of the other components of the pharmaceutically acceptable composition, its use It is contemplated to be within the scope of this invention. Some examples of materials that can serve as pharmaceutically acceptable carriers include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, whey proteins, such as human serum albumin, buffering substances, such as phosphates, glycine, sorbic acid or potassium sorbate, mixtures of partial glycerides of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium acid phosphate, potassium acid phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, wool grease, sugars, and saccharose; starches, such as cornstarch 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 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, sweetening, flavoring and perfuming agents, preservatives and antioxidants , may also be present in the composition, at the discretion of the formulator.

Uses of the pharmaceutically acceptable compounds and compositions

In another aspect, the present invention provides a method for the treatment of a CFTR-implicated condition, disease or disorder. In some embodiments, the present invention provides a method of treating a condition, disease, or disorder implicated in a deficiency of CFTR activity, the method comprising administering a composition comprising a described Form I solid state form. herein a patient, preferably a mammal, in need thereof.

A CFTR-measured disease as used herein is a selected disease of cystic fibrosis, hereditary emphysema, hereditary hemochromatosis, coagulation-fibrinolysis deficiencies, such as protein C deficiency, inherited type 1 angioedema, lipid processing deficiencies, such as familial hypercholesterolemia, chylomicronemia type 1, abetalipoproteinemia, lysosomal storage diseases, such as I-cell / pseudo-Hurler disease, mucopolysaccharidosis, Sandhof / Tay-Sachs, Crigler-Najjar type II, polndocrinopathy / hyperinsulemia, diabetes mellitus, Laron dwarfism, mileoperoxidase deficiency, primary hypoparathyroidism, melanoma, CDG glycanosis type 1, hereditary emphysema, hyperthyroidism congenital, hereditary, ACT deficiency, diabetes insipidus (DI), neurofiseal DI, neprogenic DI, Charcot-Marie dental syndrome, Perlizaeus-Merzbacher disease, neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, progressive supranuclear plasia, Pick's disease, various polyglutamine neurological disorders such as Huntington, spinocerebular ataxia, bulbar and spinal muscular atrophy, dentatorubal palidoluisian and dystrophy myotonic, as well as spongiform encephalopathies, such as hereditary Creutzfeldt-Jakob disease, Fabry disease, Straussler-Scheinker syndrome, COPD, dry eye syndrome and Sjogren's disease.

In some embodiments, the present invention provides a method for treating CFTR-mediated disease in a human, which comprises the step of administering to the human an effective amount of a composition comprising Form I described herein.

In accordance with a preferred alternative embodiment, the present invention provides a method for the treatment of cystic fibrosis in a human comprising the step of administering to the human a composition comprising the Form I described herein.

According to the invention, an effective amount of form I or a pharmaceutically acceptable composition thereof, is the effective amount for the treatment or reduction of the severity of any of the aforementioned diseases.

Form I or a pharmaceutically acceptable composition thereof can be administered using any amount and any route of administration effective for treating or lessening the severity of one or more of the aforementioned diseases.

In some embodiments, the form I described herein or a pharmaceutically acceptable composition thereof, is useful for the treatment or reduction of the severity of cystic fibrosis in patients exhibiting residual CFTR activity in the apical membrane of the respiratory epithelia and non-respiratory. The presence of residual CFTR activity on epithelial surfaces can be easily detected using methods known in the art, eg standard electrophysiological, biochemical, or histochemical techniques. These methods identify CFTR activity using in vivo or ex vivo electrophysiological techniques, measurement of IC 'concentrations of sugar or saliva, or ex vivo biochemical or histochemical techniques to monitor cell surface density. Using these methods, residual CFTR activity can be easily detected in heterozygous or homozygous patients for a variety of different mutations, including patients homozygous or heterozygous for the majority of the common mutation, ÚF5O8.

In one embodiment, Form I described herein or a pharmaceutically acceptable composition thereof is useful for treating or decreasing the severity of cystic fibrosis in patients within some genotypes exhibiting residual CFTR activity, eg, mutations of class III (damaged regulation or control), class IV mutations (altered conductance), or class V mutations (reduced synthesis) (Lee R. Choo-Kang, Pamela L. , Zeitlin, Type I, II, III, IV, and V cystic fibrosis Tansmembrane Conductance Regulator Defects and Opportunities of Therapy; Current Opinion in Pulmonary Medicine 6: 521-529, 2000). Other genotypes of patients exhibiting residual CFTR activity include patients homozygous for one of these classes or heterozygous for any other class of mutations, including class I mutations, class II mutations, or a mutation without classification.

In one embodiment, the form I described herein or a pharmaceutically acceptable composition thereof is useful for treating or decreasing the severity of cystic fibrosis in patients within some clinical phenotypes, eg, a moderate to mild clinical phenotype that it typically correlates with the amount of residual CFTR activity in the apical membrane of the epithelia. These phenotypes include patients exhibiting pancreatic insufficiency or patients diagnosed with idiopathic pancreatitis and congenital bilateral absence of vas deferens or moderate lung disease.

The exact amount required will vary from patient to patient, depending on the species, age and general condition of the patient, the severity of the infection, the particular agent, its mode of administration, and the like. The compounds of the invention are preferably formulated in unit dosage form for ease of administration and uniformity of dosage. The term "unit dosage form" as used herein refers to a physically discrete unit of agent appropriate for the patient to be treated. However, it will be understood that the total daily use of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific effective dose level for any particular patient or organism will depend on a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the specific compound used; the specific composition used; the age, body weight, general health, sex and diet of the patient; the administration time, administration route and the excretion rate of the specific compound used; the duration of treatment; drugs used in combination or coincidence with the specific compound employed and similar factors well known in the medical arts. The term patient as used herein, means an animal, preferably a mammal, and more preferably a human.

The pharmaceutically acceptable compositions of this invention can be administered to humans and other animals orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (such as by powder, ointment, or drops), orally, as an oral or nasal spray, or the like, depending the severity of the infection being treated. In some 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 the body weight of the patient daily, one or more times a day, to obtain the desired therapeutic effect.

In some embodiments, the dosage amount of form I in the unit dosage form is 100 mg to 1,000 mg. In another embodiment, the dosage amount of Form I is 200 mg to 900 mg. In another embodiment, the dosage amount of Form I is from 300 mg to 800 mg. In another embodiment, the dosage amount of Form I is from 4 00 mg to 700 mg. In another embodiment, the dosage amount of Form I is from 500 mg to 600 mg.

Injectable preparations, eg, sterile injectable aqueous or oleaginous suspensions can be formulated according to the known art using appropriate 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 as a solution in 1,3-butanediol. Acceptable vehicles and solvents that can be used include water, Ringer's solution, USP sodium chloride solution, and isotonic. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any soft fixed oil can be used, including mono- or diglycerides. Furthermore, 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-retaining 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.

Compositions for rectal or vaginal administration are preferably suppositories that can be prepared by mixing the compounds of this invention with appropriate non-irritating excipients or vehicles, such as cocoa butter, polyethylene glycol, or a suppository wax, which are solid at room temperature, but liquids at body temperature 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 such solid dosage forms, the active compound is mixed with at least one inert, pharmaceutically acceptable carrier or vehicle, such as sodium citrate or dicalcium phosphate and / or) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol and silicic acid, b) binders such as, for example, carboxymethyl cellulose, 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, some silicates and sodium carbonate, e) solution retarding agents, such as paraffin, f) absorption accelerators, such as quaternary ammonium compounds, g) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents 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 also be used as fillers in soft and hard 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 coatings, such as enteric coatings and other coatings well known in the pharmaceutical formulation art. They may optionally contain opaque agents and may also be a composition that releases only the active ingredient (s), or preferably, somewhere in the gastrointestinal tract, optionally, in a delayed manner. Examples of the coupling compositions that can be used include polymeric substances and waxes. Solid compositions of a similar type can also be used as fillers in soft and hard filled gelatin capsules using such excipients, such as lactose milk sugar, as well as high molecular weight polyethylene glycols and the like.

The active compounds can also be in the microencapsulated form with one or more excipients as mentioned above. Solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and coatings, such as enteric coatings, controlled release coatings, and other coatings well known in the art of pharmaceutical formulation. 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 in addition to 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. These may optionally contain opaque agents and may also be of a composition that they release only the active ingredients, or preferably, in a certain part of the intestinal tract, optionally , in a released manner. Examples of the coupling compositions that can be used include polymeric substances and waxes.

It will also be appreciated that the Form I described herein or a pharmaceutically acceptable composition thereof, may be used in combination with therapies, that is, Form I may be administered simultaneously with, prior to, or subsequent to, one or more desired therapeutics. or medical procedures. The particular combination of therapies (therapeutics or procedures) to be employed in a combination regimen will take into account the compatibility of the desired therapeutics and / or the procedures and the desired therapeutic effect to be achieved. It will also be appreciated that the therapies employed can achieve a desired effect for the same disorder (for example, an inventive compound can be administered simultaneously with another agent used to treat the same disorder) or can achieve different effects (for example, control of any adverse effects ). 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 being treated.

In one embodiment, the additional agent is selected from a mucolytic agent, bronchodilator, an antibiotic, an anti-infective agent, an anti-inflammatory agent, a CFTR modulator other than a compound of the present invention, or a nutritional agent.

In other embodiments, the additional agent is a compound selected from gentamicin, curcumin, cyclophosphamide, 4-phenylbutyrate, miglustat, felodipine, nimodipine, Philoxin B, geniestein, Apigenin, cAMP / cGMP modulators, such as rolipram, sildenafil, milrinone, tadalafil , amrinone, isoproterenol, albuterol and almeterol, desoxispergualin, HSP 90 inhibitors, HSP 70 inhibitors, proteasome inhibitors, such as epoxomycin, lactacystin, etc.

In another embodiment, the additional agent is a compound described in WO 2004028480, WO 2004110352, WO 2005094374, WO 2005120497 or WO 2006101740.

In another embodiment, the additional agent is a benzo (c) quinolizinium derivative exhibiting CFTR modulating activity or a benzopyran derivative exhibiting CFTR modulating activity.

In another embodiment, the additional agent is a compound described in US7202262, US6992096, US20060148864,

US20060148863, US20060035943, US20050164973, W02006110483, W02006044456, W02006044682, W02006044505, W02006044503, W02006044502 or W02004091502.

In another embodiment, the additional agent is a compound described in W02004080972, W02004111014, W02005035514,

W02005049018, W02006002421, W02006099256, WO2006127588 or

W02007044560.

In another embodiment, the additional agent is selected from the compounds described in US Patent Application Serial No. 11 / 165,818, published as US Published Patent Application No. 2006/0074075, filed June 24, 2005, and which is incorporated herein by reference in its entirety. In another embodiment, the additional agent is N- (5-hydroxy-2,4-diter-butyl-phenyl) -4-oxo-lH-quinolin-3carboxamide. These combinations are useful for the treatment of the diseases described herein including cystic fibrosis. These combinations are also useful in the kits described herein.

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

Form I described herein or a pharmaceutically acceptable composition thereof can also be incorporated into compositions for coating an implantable medical device, such as prostheses, artificial valves, vascular grafts, stents, and catheters. Therefore, the present invention, in another aspect, includes a composition for the coating of an implantable device comprising the Form I described herein or a pharmaceutically acceptable composition thereof, and in the classes and subclasses herein, and an appropriate vehicle for coating such an implantable device. In yet another aspect, the present invention includes an implantable device coated with a composition comprising the Form I described herein or a pharmaceutically acceptable composition thereof, and an appropriate vehicle for coating such an implantable device. Appropriate coatings and general preparation of coated implantable devices are described in US Patents 6,099,562; 5,886,026 and 5,304,121. The coatings are typically biocompatible polymeric materials, such as hydrogel polymer, polymethyldisiloxane, polycaprolactone, polyethylene glycol, polylactic acid, ethylene vinyl acetate, and mixtures thereof. The coatings can optionally be covered by an appropriate topcoat of fluorosilicone, polysaccharides, polyethylene glycol, phospholipids, or combinations thereof to impart controlled release characteristics to the composition.

For this the invention described herein can be more fully understood by setting the following examples. It should be understood that these examples are for illustrative purposes only and are not intended to be limiting of this invention in any way.

EXAMPLES

Methods and Materials

Differential scanning calorimetry (DSC)

Differential scanning calorimetry (DSC) data from Form I was collected using a DSC Q100 V9.6 Build 290 (TA Instruments, New Castle, DE). The temperature was calibrated with indium and the heat capacity was calibrated with sapphire. The 3-6 mg samples were weighed into aluminum containers that were corrugated using 1-hole lids. Samples were scanned from 25 ° C to 350 ° C at a heating rate of 1.0 ° C / min and with a 50 ml / min nitrogen gas purge. Data was collected by Thermal Advantage Q Series ™ software version 2.2.0.248 and analyzed by Universal Analysis software version 4. ID (TA Instruments, New Castle, DE). The reported numbers represent simple analyzes.

XRPD (X-ray Spray Diffraction)

X-ray diffraction (XRD) data from Form 1 was collected on a Bruker D8 DISCOVER spray diffractometer with a two-dimensional HI-STAR detector and a flat graphite monochromator. A Cu tube sealed with Ka radiation at 40 kV, 35 mA was used. The samples were placed on zero-bottom silicon contact plates at 25 ° C. For each sample, two data frames were collected at 120 seconds each at 2 different angles θ₂: 8^or and 26 °. Data was integrated with GADDS software and pooled with DIFFRACT software^plusEVE. Uncertainties for reported peak positions are + 0.2 degrees.

Vitride® (sodium bis (2-methoxyethoxy) aluminum hydride [or NaAlH₂ (OCH2CH2OCH3) ₂], 65% by weight solution in toluene) was purchased from Aldrich Chemicals.

2,2-Difluoro-1,3-benzodioxol-5-carboxylic acid was purchased from Saltigo (an affiliation with the Lanxess Corporation).

Wherever in the present application where a compound name cannot correctly describe the structure of the compound, the structure overlaps the name and takes precedence.

Synthesis of 3- (6- (1- (2,2-Difluorobenzo [d] [1,31-dioxol5-yl) cyclopropancarboxamido) -3-methylpyridin-2-yl) benzoic acid · HC1

Portion of acid chloride

Synthesis of (2,2-difluoro-1,3-benzodioxol-5-yl) -methanol.

one. Vitride (2 equiv)

PhCH₃ (10 vol)

2. NaOH 10% aq (w / w) (4 equiv)

86-92% yield

Commercially available 2,2-difluoro-1,3-benzodioxol-5-carboxylic acid (1.0 eq) is suspended in toluene (10 vol). Vitride® (2 eq) is added via an addition funnel at a rate to maintain the temperature at 15-25 ° C. At the end of the addition the temperature is increased to 4 0 ° C for 2 h then 10% (w / w) aqueous NaOH (4.0 eq) is carefully added by means of an addition funnel keeping the temperature at 40-50 ° C. After stirring for an additional 30 minutes, the layers are allowed to separate at 40 ° C. The organic phase is cooled to 20 ° C then washed with water (2 x 1.5 vol), dried (Na₂SW₄), filter and concentrate to give crude (2,2-difluoro-1,3-benzodioxol-5-yl) -methanol which is used directly in the next step.

Synthesis of 5-chloromethyl-2,2-difluoro-1,3-benzodioxol.

<img file="MX367154B_D0015.tif" />

one. SOC1, (1.5 cquiv)

DMAP (0.01 equiv)

MTBE (5 vol)

2. water (4 vol)

<img file="MX367154B_D0016.tif" />

Yield 82-100 (2,2-Difluoro-1,3-benzodioxol-5-yl) -methanol (1.0 eq) is dissolved in MTBE (5 vol). A catalytic amount of DMAP (1 mol%) is added and SOC1 is added₂ (1.2 eq) by means of an addition funnel. SOC1 is added₂at a rate to maintain the temperature in the reactor at 15-25 ° C. The temperature is increased to 30 ° C for 1 hour then it is cooled to 20 ° C, then water (4 vol) is added by means of an addition funnel keeping the temperature below 30 ° C. After stirring for an additional 30 minutes, the layers are allowed to separate. The organic layer is stirred and 10% (w / v) aqueous NaOH (4.4 vol) is added. After stirring for 15-20 minutes, the layers are allowed to separate. The organic phase is then dried (Na₂SW₄), filtered and concentrated to give crude 5-chloromethi-1,2,2-difluoro-1,3-benzodioxol which is used directly in the next step.

Synthesis of (2,2-difluoro-1,3-benzodioxol-5-yl) -acetonitrile.

one. NaCN (1.4 equiv) DMSO (3 vol) 30-40 degrees C

2. water (6 vol) ^F_V^ORY ^ MTBE (4 vol) ^FxZ ° 'TÍ ^ |

95-100 performance

A solution of 5-chloromethyl-2,2-difluoro-1,3benzodioxol (1 eq) in DMSO (1.25 vol) is added to a suspension of NaCN (1.4 eq) in DMSO (3 vol) keeping the temperature between 30-40 ° C. The mixture is stirred for 1 hour 10 then water (6 vol) is added followed by MTBE (4 vol). After stirring for 30 minutes, the layers are separated. The aqueous layer is extracted with MTBE (1.8 vol). The combined organic layers are washed with water (1.8 vol), dried (Na₂SW₄), filtered and concentrated to give crude (2,2-difluoro15 1,3-benzodioxol-5-yl) -acetonitrile (95%) which is used directly in the next step.

Synthesis of (2,2-difluoro-1,3-benzodioxol-5-yl) cyclopropancarbonitrile.

<img file="MX367154B_D0017.tif" />

<img file="MX367154B_D0018.tif" />

0 1-bromo-2-chloroethane (1.5 equiv)

50% KOH (5.0 equiv)

Oct₄NBr (0.02 equiv) 70 ° C 88-100 performance

A mixture of (2,2-difluoro-1,3-benzodioxol-5-yl) acetonitrile (1.0 eq), 50% by weight aqueous KOH (5.0 eq), 125 bromo-2-chloroethane (1.5 eq) and OCt₄NBr (0.02 eq) is heated to

70 ° C for 1 h. The reaction mixture is cooled after working up with MTBE and water. The organic phase is washed with water and brine, then the solvent is removed to give (2,2difluoro-1,3-benzodioxol-5-yl) -cyclopropancarbonitrile.

<img file="MX367154B_D0019.tif" />

<img file="MX367154B_D0020.tif" />

Synthesis of 1- (2,2-difluoro-1,3-benzodioxol-5-yl) cyclopropancarboxylic acid.

one. 6M NaOH (8 equiv) EtOH (5 VOl), 80 ° C

2. MTBE (10 vol) dicyclohexylamine (1 equiv)

CN

3. MTBE (10 vol) citric acid 10% ac (8 vol)

69% yield (2,2-difluoro-1,3-benzodioxol-5-yl) cyclopropancarbonitrile is hydrolyzed using 6M NaOH (8 equivalents) in ethanol (5 vol) at 8 0 ° C overnight. The mixture is cooled to room temperature and ethanol is evaporated in vacuo. The residue is placed in water and MTBE, 1M HCI is added and the layers are separated. The MTBE layer is then treated with dicyclohexylamine (0.97 equivalents). The suspension is cooled to 0 ° C, filtered and washed with heptane to give the corresponding DCHA salt. The salt is placed in MTBE and 10% citric acid and stirred until all solids have dissolved. The layers are separated and the MTBE layer is washed with water and brine. The solvent is changed to heptane followed by filtration to give 1- (2,2-difluoro-1,3-benzodioxol-5-yl) -cyclopropancarboxylic acid after drying in a vacuum oven at 50 ° C overnight.

Synthesis of 1- (2,2-difluoro-1,3-benzodioxol-5-yl) cyclopropancarbonyl chloride.

SOC1₂,

P11CH₃, ^FV ° 'll n 60 ° C ⁰

Λ JU JL ---------------- ► z \ II J 11 fo oh f ο '^^{ί! ί}7ς'α

1- (2,2-Difluoro-1,3-benzodioxol-5-yl) cyclopropancarboxylic acid (1.2 eq) is suspended in toluene (2.5 vol) and the mixture is heated to 60 ° C. SOCI2 (1.4 eq) is added by means of an addition funnel. Toluene and SOC1₂ they are distilled from the reaction mixture after 30 minutes. Additional toluene (2.5 vol) is added and it is distilled again.

Amine portion

Synthesis of tert-butyl-3- (3-methylpyridin-2-yl) benzoate.

<img file="MX367154B_D0021.tif" />

<img file="MX367154B_D0022.tif" />

one. toluene. K ^ COj 2M Pd (dppf) Ch, 80 ° C

2. MsOH ac.

3. NaOH ac.

<img file="MX367154B_D0023.tif" />

2-Bromo-3-methylpyridine (1.0 eq) is dissolved in toluene (12 vol). K is added₂CO₃ (4.8 eq) followed by water (3.5 vol) and the mixture is heated to 65 ° C under a stream of N₂for 1 hour. Then 3- (tbutoxycarbonyl) phynylboronic acid (1.05 eq) and Pd (dppf) C1 are added₂-CH₂C1₂ (0.015 eq) and the mixture is heated to 80 ° C. After 2 hours, the heat is removed, water (3.5 vol) is added and the layers are allowed to separate. The organic phase is then washed with water (3.5 vol) and extracted with 10% aqueous methanesulfonic acid (MsOH 2 eq, 7.7 vol). The aqueous phase is basified with 50% aqueous NaOH (2 eq) and extracted with EtOAc (8 vol).

The organic layer is concentrated to give crude terbutyl-3- (3-methylpyridin-2-yl) benzoate (82%) which is used directly in the next step.

Synthesis of 2- (3- (tert-butoxycarbonyl) phenyl) -3-methylpyridine-oxide.

<img file="MX367154B_D0024.tif" />

CC ^ tBu urea-hydrogen peroxide phthalic anhydride EtOAc, water

<img file="MX367154B_D0025.tif" />

Tert-Butyl-3- (3-methylpyridin-2-yl) benzoate (1.0 eq) is dissolved in EtOAc (6 vol). Water (0.3 vol) is added followed by urea-hydrogen peroxide (3 eq). Phthalic anhydride (3 eq) is added portion by portion as a solid to keep the temperature in the reactor below 45 ° C. After completing the addition of phthalic anhydride, the mixture is heated to 45 ° C. After stirring for an additional 4 hours, the heat is removed. Na is added₂SW₃ aqueous 10% w / w (1.5 eq) by means of an addition funnel. After completing the addition of Na₂SW₃, the mixture is stirred for an additional 30 minutes and the layers are separated. The organic layer is stirred and Na is added₂C03 aqueous 10% w / w (2 eq). After stirring for 30 minutes, the layers are allowed to separate. The organic phase is washed with 13% w / v aqueous NaCl. The organic phase is then filtered and concentrated to give crude 2 (3- (tert-butoxycarbonyl) phenyl) -3-methylpyridin-l-oxide (95%) which is used directly in the next step.

Synthesis of tert-butyl-3- (6-amino-3-methylpyridin2-yl) benzoate.

N

one. More₂O, py, MeCN, 70 ° c

2. ethanolamine h₂n''n

CO₂© u

CO₂®u

A solution of 2- (3- (tert-butoxycarbonyl) phenyl) -3methylpyridin-1-oxide (1 eq) and pyridine (4 eq) in MeCN (8 vol) is heated to 70 ° C. A solution of methanesulfonic anhydride (1.5 eq) in MeCN (2 vol) is added over 50 minutes using an addition funnel, keeping the temperature below 75 ° C. The mixture is stirred for an additional 0.5 hours after the addition is complete. The mixture is then allowed to cool to ambient. Ethanolamine (10 eq) is added via an addition funnel. After stirring for 2 hours, water (6 vol) is added and the mixture is cooled to 10 ° C. After stirring for 3 hours NLT, the solid is collected by filtration and washed with water (3 vol), MeCN / water 2: 1 (3 vol) and MeCN (2 x 1.5 vol). The solid is dried at constant weight (&lt;1% difference) in a vacuum oven at 50 ° C with a slight spillage of N₂ to give tert-butyl-3 (6-amino-3-methylpyridin-2-yl) benzoate as a red-yellow solid (53% yield).

Synthesis of benzoate from

3- (6- (1- (2,2difluorobenzo [d] [1,3] dioxol-5-yl) cyclopropancarboxamido) -3methylpyridin-2-yl) -t-butyl.

<img file="MX367154B_D0026.tif" />

CO₂tBu

<img file="MX367154B_D0027.tif" />

The crude acid chloride is dissolved in toluene (2.5 vol based on the acid chloride) and added via an addition funnel to a mixture of tert-butyl-3- (6-amino-3-methylpyridin-2-yl benzoate). ) (1 eq), dimethylaminopyridine (DMAP, 0.02 eq) and triethylamine (3.0 eq) in toluene (4 vol based on tert-butyl-3- (6-amino-3-methylpyridin-2yl benzoate). After 2 hours, water (4 vol based on tert-butyl-3- (6-amino-3-methylpyridin ~ 2-yl)) is added to the reaction mixture. After stirring for 30 minutes, the layers are separated. The organic phase is then filtered and concentrated to give a thick benzoate oil of 3- (6- (1- (2,2difluorobenzo [d] [1,3] dioxol-5-yl) cyclopropancarboxamido) -3methylpyridin-2- il) -t-butyl (quantitative crude yield). MeCN (3 vol based on the crude product) is added and distilled until crystallization occurs. Water (2 vol based on the crude product) is added and the mixture is stirred for 2 h. The solid is collected by filtration, washed with (by volume) 1: 1 MeCN / water (2x1 vol based on the crude product) and partially dried on the filter under vacuum. The solid is dried at constant weight (&lt;1% difference) in a vacuum oven at 60 ° C with a slight spillage of N₂ to give 3- (6- (1- (2,2difluorobenzo [d] [1,3] dioxol-5-yl) cyclopropancarboxamido) -3-methylpyridin-2-yl) -t-butyl benzoate as a brown solid.

Synthesis of 3- (6- (1- (2,2-Difluorobenzo [d] [1,3] dioxol5-yl) cyclopropancarboxamido) -3-methylpyridin-2-yl) benzoic acid * HC1 salt.

<img file="MX367154B_D0028.tif" />

• HC1

To a suspension of 3- (6- (1- (2,248 difluorobenzo [d] [1,3] dioxol-5-yl) cyclopropancarboxamido) -3methylpyridin-2-yl) -t-butyl (1.0 eq) benzoate in MeCN (3.0 vol) water is added (0.83 vol) followed by concentrated aqueous HC1 (0.83 vol). The mixture is heated to 45 ± 5 ° C. After stirring for 24 to 48 hours the reaction is complete and the mixture is allowed to cool to room. Water (1.33 vol) is added and the mixture is stirred. The solid is collected by filtration, washed with water (2 x 0.3 vol), and partially dried on the filter under vacuum. The solid is dried at constant weight (&lt;1% difference) in a vacuum oven at 60 ° C with a slight spillage of N₂ to give 3- (6- (1- (2,2difluorobenzo [d] [1,3] dioxol-5-yl) cyclopropancarboxamido) -3methylpyridin-2-yl) benzoic acid · HC1 as an almost white solid.

Synthesis of 3- (6- (1- (2,2-Difluorobenzo [d] [1,3] dioxol5-yl) cyclopropancarboxamido) -3-methylpyridin-2-yl) benzoic acid (Form I).

NN

H

HCl suspension in water ^FX °

FO ^ xCO₂H

Acid suspension

3- (6- (1- (2,2 Form I dif luorobenzo [d] [1,3] dioxol-5-yl) cyclopropancarboxamido) -3methylpyridin-2-yl) benzoic · HC1 (1 eq) in water (10 vol ) is stirred at room temperature. A sample is taken after shaking for 24 hours. The sample is filtered and the solid is washed with water (2x). The solid sample is prepared for DSC analysis. When DSC analysis indicates complete conversion for Form I, the solid is collected by filtration, washed with water (2 x 1.0 vol), and partially dried on the filter under vacuum. The solid is dried at constant weight (&lt;1% difference) in a vacuum oven at 60 ° C with a slight spillage of Na to give Form I as an almost white solid (98% yield). 'Ή NMR (400 MHz, DMSOd6) 9.14 (s, 1H), 7.99-7.93 (m, 3H), 7.80-7.78 (m, 1H), 7.74-7.72 (m, 1H), 7.60-7.55 (m, 2H ), 7.41-7.33 (m, 2H), 2.24 (s, 3H), 1,531.51 (m, 2H), 1.19-1.17 (m, 2H).

Synthesis of 3 - (6- (1- (2,2-Difluorobenzo [d] [1,3] dioxol5-yl) cyclopropancarboxamido) -3-methylpyridin-2-yl) benzoic acid (Form I) using water and base.

<img file="MX367154B_D0029.tif" />

one. H₂0, 50% NaOH

2. HC1 conc.

60-90 ° C

<img file="MX367154B_D0030.tif" />

Form I

To a suspension of 3- (6- (1- (2,2difluorobenzo [d] [1,3] dioxol-5 i1) ci clopropancarboxami do) - 3-me tilpyridin-2yl) benzoic acid · HC1 (1 eq) in Water (10 vol) stirred at room temperature, 50% w / w (2.5 eq) aqueous NaOH is added. The mixture is stirred for NLT 15 minutes or until the solution is homogeneous. Concentrated HC1 (4 eq) is added to crystallize Form I. The mixture is heated to 60 ° C or 90 ° C if necessary to reduce the level of the tbutiIbenzoate ester. The mixture is heated until HPLC analysis indicates 0.8% NMT (AUC) ester tbutiIbenzoate. The mixture is then cooled to room temperature and the solid is collected by filtration, washed with water (3 x 3.4 vol), and partially dried on the filter under vacuum. The solid is dried at constant weight (&lt;1% difference) in a vacuum oven at 60 ° C with a slight spillage of N₂ to give Form I as an almost white solid (97% yield).

Synthesis of 3- (6- (1- (2,2-Difluorobenzo [d] [1,3] dioxol5-yl) cyclopropancarboxamido) -3-methylpyridin-2-yl) benzoic acid (Form I) directly from benzoate.

NN

H

CO_ztBu 70 C

one. formic acid

2. Water

NN

H

Form I

A solution of 3- (6- (1- (2,2-difluorobenzo [d] [1,3] dioxol-5 i1) cyclopropancarboxamido) - 3-methypyridin-2-i1) -tbutyl (1.0 eq) benzoate in formic acid (3.0 vol) is heated to 70 + 10 ° C. The reaction is continued until complete (NMT 1.0% AUC of 3- (6- (1- (2,2-difluorobenzo [d] [1,3] dioxol-5i1) cyclopropancarboxamido) -3-methypyridin-2-i1 benzoate) ) -1butyl) or NMT heating 8 h. The mixture is allowed to cool to room. Water is added to the solution (6 vol) heated to 50 ° C and the mixture is stirred. The mixture is then heated to 70 + 10 ° C until the benzoate level of 3- (6- (1- (2,2difluorobenzo [d] [1,3] dioxol-5 25 yl) cyclopropanearboxamido) - 3-methIpiridin -2-yl) -1 butyl is NMT 0.8% (AUC). The solid is collected by filtration, washed with water (2x3 vol), and partially dried on the filter under vacuum. The solid is dried at constant weight (&lt;1% difference) in a vacuum oven at 60 ° C with a slight spillage of N₂ to give compound 1 in Form I as an almost white solid.

A calculated X-ray diffraction pattern of a single crystal structure of compound 1 in Form I is shown in Figure 1. Table 1 lists the calculated peaks for Figure 1.

Table 1.

<td>Peak classification</td><td>Angle 2Θ [degrees]</td><td>Relative intensity [%]</td>

<td> 11</td><td> 14.41</td><td> 48.2</td>

<td> 8</td><td> 14.64</td><td> 58.8</td>

<td> 1</td><td> 15.23</td><td> 100.0</td>

<td> 2</td><td> 16.11</td><td> 94.7</td>

<td> 3</td><td> 17.67</td><td> 81.9</td>

<td> 7</td><td> 19.32</td><td> 61.3</td>

<td> 4</td><td> 21.67</td><td> 76.5</td>

<td> 5</td><td> 23.40</td><td> 68.7</td>

<td> 9</td><td> 23.99</td><td> 50.8</td>

<td> 6</td><td> 26.10</td><td> 67.4</td>

<td> 10</td><td> 28.54</td><td> 50.1</td>

An actual X-ray spray diffraction pattern of Compound 1 in Form I is shown in Figure 2. Table 2 lists the actual peaks for Figure 2.

Table 2.

<td>Classification of peak</td><td>Angle 2Θ [degrees]</td><td>Relative intensity [%]</td>

<td> 7</td><td> 7.83</td><td> 37.7</td>

<td> 3</td><td> 14.51</td><td> 74.9</td>

<td> 4</td><td> 14.78</td><td> 73.5</td>

<td> 1</td><td> 15.39</td><td> 100.0</td>

<td> 2</td><td> 16.26</td><td> 75.6</td>

<td> 6</td><td> 16.62</td><td> 42.6</td>

<td> 5</td><td> 17.81</td><td> 70.9</td>

<td> 9</td><td> 21.59</td><td> 36.6</td>

<td> 10</td><td> 23.32</td><td> 34.8</td>

<td> 11</td><td> 24.93</td><td> 26.4</td>

<td> 8</td><td> 25.99</td><td> 36.9</td>

An overlay of a calculated X-ray diffraction pattern of a single crystal structure of Compound 1 in Form I, and an actual X-ray spray diffraction pattern of Compound 1 in Form I is shown in Figure 3. The Overlap shows good agreement between the calculated and actual peak positions, the difference being only about 0.15 degrees.

The DSC trace of Compound 1 in Form I is shown in Figure 4. Melting for Compound 1 in Form I occurs at approximately 204 ° C.

The conformational images of Compound 1 in Form I based on crystalline X-ray analysis is shown in Figures 5-8. Figures 6-8 show the hydrogen bond between the carboxylic acid groups of a dimer and the resulting stack that occurs in the crystal. The crystal structure reveals a dense packing of the molecules. Compound 1 in Form I is monoclinic, P2x / n, with the following unit cell dimensions: a = 4.9626 (7) Á, b = 12.299 (2) Á, c = 33.075 (4) Á, β = 93.938 ( 9) °, V = 2014.0 A³, Z = 4. The density of Compound 1 in Form I calculated from the structural data is 1,492 g / cm.³ at 100 K.

The HRMN spectra of Compound 1 are shown in Figures 9-11 (Figures 9 and 10 represent Compound 1 in Form I in a suspension of 5 0 mg / mL, methyl cellulose 0.5-polysorbate 80, and Figure 11 depicts compound 1 as an HCI salt).

Table 3 below mentions additional analytical data for compound 1.

Table 3.

<td>Comp. No.</td><td>LC / MS M + l</td><td>LC / RT min</td><td>NMR</td>

<td> 1</td><td> 453.3</td><td> 1.93</td><td>1H NMR (400 MHz, DMSO-d6) 9.14 (s, 1H), 7.99-7.93 (m, 3H), 7.80-7.78 (m, 1H), 7.74-7.72 (m, 1H), 7.60-7.55 (m, 2H), 7.41-7.33 (m, 2H), 2.24 (s, 3H), 1.53- 1.51 (m, 2H), 1.19-1.17 (m, 2H).</td>

TESTS

Tests to detect and measure the correction properties of AF508-CFTR of compounds

Potential membrane optical methods to test the AF508-CFTR modulating properties of compounds

The optical membrane potential used the voltage-sensitive FRET detectors described by González and Tsien (See González, JE and RY Tsien (1995) Voltage sensing by fluorescence resonance energy transfer in single cells Biophys J 69 (4): 1272-80, and González, JE and RY Tsien (1997) Improved indicators of cell membrane potential that use fluorescence resonance energy transfer Chem Biol 4 (4): 269-77) in combination with instrumentation to measure changes in fluorescence such as voltage probe / ion probe (VIPR) ( see, González, JE, K. Oades, et al. (1999)

Cell-based assays and instrumentation for screening ionchannel targets Drug Discov Today 4 (9): 431-439).

These voltage sensitive tests are based on the change in fluorescence resonant energy transfer (FRET) between the soluble membrane voltage sensitive dye, DiSBAC₂(3) and a fluorescent phospholipid, CC2-DMPE, which binds to the outer leaflet of the plasma membrane and acts as a FRET donor. Changes in membrane potential (V_m) cause DiSBAC₂(3) Negatively charged redistributes across the plasma membrane and therefore the amount of energy transfer of CC2-DMPE changes. Changes in fluorescence emission were monitored using VIPR ™ II, which is an integrated liquid handler and fluorescent detector designed to drive cell-based sieves into 96- or 384-well microtiter plates.

one. Identification of correction compounds

To identify small molecules that correct the traffic defect associated with AF508-CFTR; A single-addition HTS test format was developed. Cells were incubated in serum-free medium for 16 hours at 37 ° C in the presence or absence (negative control) of the test compound. As a positive control, cells plated in 384-well plates were incubated for 16 hours at 27 ° C to correct the temperature of AF508-CFTR. Cells were subsequently rinsed 3X with Krebs Ringer's solution and loaded with voltage sensitive dyes. To activate AF508-CFTR, 10 µΜ forskolin and the CFTR enhancer, genistein (20 µΜ), along with Cl 'free medium, were added to each well. The addition of CI 'free medium promoted Cl' flux in response to AF508-CFTR activation and the resulting membrane depolarization was monitored optically using FRET-based voltage detector dyes.

2. Identification of enhancer compounds

To identify the enhancers of AF508-CFTR, a double-added HTS test format was developed. During the first addition, a Cl 'free medium with or without the test compound was added to each well. After 22 seconds, a second addition of C1 'free medium containing 2-10 µΜ forskolin was added to activate AF508-CFTR. The concentration of extracellular Cl 'after both additions was 2 8 mM, which promoted the flow of Cl' in response to activation of AF508-CFTR and the resulting membrane depolarization was optically monitored using the voltage detector dyes at FRET base.

3. Solutions

Bath solution # 1: (in mM) NaCl 160, KC1 4.5, CaCl₂ 2,

MgCl₂ 1, HEPES 10, pH 7.4 with NaOH.

Chloride-free bath solution: Chloride salts in bath solution # 1 are replaced with gluconate salts.

CC2-DMPE: Prepared as a 10mM standard solution in DI4SO and stored at -2 0 ° C.

DiSBAC₂(3): Prepared as a 10mM standard in DMSO and stored at -20 ° C.

Four. Cell culture NIH3T3 mouse fibroblasts stably expressing AF508-CFTR are used for optical measurements of membrane potential. Cells are kept at 37 ° C in C0₂ 5% and 90% humidity in Dulbecco's modified Eagle's medium supplemented with 2 mM glutamine, 10% fetal bovine serum, 1 X NEAA, β-ΜΕ, 1 X pen / strep and 25 mM HEPES in culture flasks of 175 cm² . For all optical tests, cells are seeded at 30,000 / well in 384-well matrigel-coated plates and cultured for 2 hours at 37 ° C before culturing at 27 ° C for 24 hours for the enhancer test. For correction tests, cells are grown at 27 ° C or 37 ° C with and without compounds for 16-24 hours.

Electrophysiological tests to test the AF508-CFTR modulating properties of compounds

one. Using the camera test

Chamber experiments were performed on polarized epithelial cells expressing AF508-CFTR to further characterize the AF508CFTR modulators identified in the optical tests. Epithelial cells frt^A508‘^cftr grown in Costar Snapwell cell culture inserts were mounted in a Ussing chamber (Physiologic Instruments, Inc., San Diego, CA) and the monolayers were continuously short-circuited using a voltage clamp system (Department of Bioengineering, University of lowa , IA, and, Physiologic Instruments, Inc., San Diego, CA). Transepithelial resistance was measured by applying a 2 mV pulse. Under these conditions, the FRT epithelia demonstrated resistance of 4 ΚΩ / cm² or more. The solutions were kept at 27 ° C and bubbled with air. Electrode drift potential and fluid resistance were corrected using a cell-free insert. Under these conditions, the current reflects the Cl 'flux through AF508-CFTR expressed on the apical membrane. The I_sc it was acquired digitally using an MP100A-CE interface and AcqKnowledge software (v3.2.6; BIOPAC Systems, Santa Barbara, CA).

2. Identification of correction compounds

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

As observed in the other cell types, incubation at low temperatures of FRT cells stably expressing AF508-CFTR increases the functional density of CFTR in the plasma membrane. To determine the activity of the correction compounds, the cells were incubated with 10 µΜ of the test compound for 24 hours at 37 ° C and subsequently washed 3X prior to registration. The I_sc cAMP and genistein-mediated cells treated with the compound were normalized to the 27 ° C and 37 ° C controls and expressed as a percentage activity. Preincubation of cells with the correction compound significantly increased I_sc cAMP and genistein mediated compared to controls at 37 ° C.

3. Identification of enhancer compounds

The typical protocol used a C1 'concentration gradient from basolateral to apical membrane. To adjust this gradient, normal Ringer's solution was used in the basolateral membrane and it was permeabilized with nystatin (360 pg / ml), while apical NaCl was replaced by equimolar sodium gluconate (titrated to pH 7.4 with NaOH) to give a gradient of large C1 'concentration through the epithelium. All experiments were performed 30 minutes after nystatin permeabilization. Forskolin (10 µΜ) was added and all test compounds were added to both sides of the cell culture inserts. The efficacy of the putative AF508-CFTR enhancers was compared to that of the known enhancer, genistein.

Four. Solutions

Basolateral solution (in mM): NaCl (135), CaCl₂ (1.2), MgCl₂(1.2), K₂HPO₄ (2.4), KHPO₄ (0.6), N-2-hydroxyethylpiperazin-N acid¹-2-ethanesulfonic (HEPES) (10) and dextrose (10). The solution was titrated to pH 7.4 with NaOH.

Apical solution (in mM): The same as the basolateral solution with NaCl replaced with Na gluconate (135).

5. Cell culture

Fisher rat epithelial cells (FRT) expressing

AF508-CFTR (FRT^{áF508 CFTR}) were used for the Ussing chamber experiments for the putative AF508-CFTR modulators identified from the optical tests. Cells were grown in Costar Snapwell cell culture inserts and grown for five days at 37 ° C and 5% CO₂ in Coon's modified Ham's F-12 medium supplemented with 5% fetal calf serum, 100 U / ml penicillin and 100 pg / ml streptomycin. Before use to characterize the enhancing activity of the compounds, the cells were incubated at 27 ° C for 16-48 hours to correct the AF508-CFTR. To determine the activity of the correction compounds, cells were incubated at 27 ° C or 37 ° C with and without the compounds for 24 hours.

6. Complete cellular records

The stream of macroscopic AF508-CFTR (Ilesos) in NIH3T3 cells corrected with the test compound stably expressing AF508-CFTR were monitored using the full, perforated patch cell registry. Briefly, Ilesos θθ voltage clamp recordings were performed at room temperature using an Axopatch 200B patch clamp amplifier (Axon Instruments Inc., Foster City, CA). All records were acquired at a sampling rate of 10 kHz and filtered at a low pass at 1 kHz. The pipettes had a resistance of 5-6 ΜΩ when filled with the intracellular solution. Under these recording conditions, the calculated reversal potential for Cl (Ea) at room temperature was -28 mV. All records had a sealing resistance&gt; 20 GO and a series resistance &lt;15 ΜΩ. 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 μΐ of saline and was continuously perfused at a rate of 2 ml / min using a gravity powered perfusion system.

7. Identification of correction compounds

To determine the activity of correction compounds to increase the density of functional AF508-CFTR on the plasma membrane, the perforated patch recording techniques described above were used to measure current density after 24 hours of treatment with the compounds of correction. To fully activate AF508-CFTR, 10 µΜ forskolin and 20 µΜ genistein were added to the cells. Under these recording conditions, the current density after 24 hours of incubation at 2 7 ° C was higher than that observed after 24 hours of incubation at 37 ° C. These results are consistent with the known effects of low temperature incubation on the density of AF508-CFTR in the plasma membrane. To determine the effects of the correction compounds on the CFTR current density, cells were incubated with 10 µΜ of the test compound for 24 hours at 37 ° C and the current density was compared to controls at 27 ° C and 37 ° C (% activity). Before registration, cells were washed 3X with extracellular recording medium to remove any remaining test compound. Pre-incubation with 10 µΜ of the correction compounds significantly increased the cAMP and genistein dependent current compared to controls at 37 ° C.

8. Identification of enhancer compounds

The ability of AF508-CFTR enhancers to increase the macroscopic Cl 'current of AF508-CFTR (Iafsos) θη NIH3T3 cells stably expressing AF508-CFTR was also investigated using perforated patch recording techniques. The identified enhancers from the optical tests elicited a dose dependent increase in I_AFso5 with similar power and efficacy observed in optical tests. In all examined cells, the reversal potential before and during the enhancer application was around -30 mV, which is the E_Ci calculated (-28 mV).

9. Solutions

Intracellular solution (in mM): Cs (90) aspartate, CsCl (50), MgCl₂ (1), HEPES (10) and 240 pg / ml amphotericinB (pH adjusted to 7.35 with CsOH).

Extracellular solution (in mM): N-methyl-D-glucamine (NMDG) -C1 (150), MgCl₂ (2), CaCl₂ (2), HEPES (10) (pH adjusted to 7.35 with HC1).

10. Cell culture

NIH3T3 mouse fibroblasts stably expressing AF508-CFTR are used for whole cell recordings. Cells are maintained at 37 ° C in 5% CO₂ and 90% humidity in Dulbecco's modified Eagle medium supplemented with 2 mM glutamine, 10% fetal bovine serum, NEAA IX, βME, pen / strep IX and 5 mM HEPES 2 in 175 cm² of culture flasks. For complete cell records, 2,500-5,000 cells were seeded on poly-L-lysine coated glass coverslips and cultured for 24-48 hours at 27 ° C before use to test for enhancer activity; and incubated with or without the correction compound at 37 ° C to measure the activity of the correctors.

eleven. Simple channel records

Single channel activities of temperature corrected AF508-CFTR stably expressed in cells

Τ3Τ3 and the activities of the enhancing compounds were observed using an inside-outside cut membrane patch. Briefly, single-channel activity voltage clamp recordings were performed at room temperature with an Axopatch 200B clamp-clip amplifier (Axon Instruments Inc.). All records were acquired at a sampling rate of 10 kHz and filtered at a low pass at 400 Hz. The patch pipettes were made from Corning Kovar Sealing # 7052 glass (World Precision Instruments, Inc., Sarasota, FL) and had a resistance of 5-8 ΜΩ when filled with the extracellular solution. AF508-CFTR was activated after excision, adding 1 mM and 75 nM Mg-ATP of the catalytic subunit of cAMP-dependent protein kinase (PKA; Promega Corp. Madison, WI). After stabilizing channel activity, the patch was perfused using a gravity activated microperfusion system. The influx was placed adjacent to the patch, resulting in complete exchange of the solution within 1-2 seconds. To maintain AF508-CFTR activity during rapid infusion, non-specific phosphatase inhibitor F (10mM NaF) was added to the bath solution. Under these recording conditions, channel activity remained constant throughout the duration of the patch registration (up to 60 minutes). The currents produced by the positive charge moving from the intra- to extracellular solutions (anions moving in the opposite direction) are shown as positive currents. Pipette potential (V_p) was kept at 80 mV.

Channel activity was analyzed from the membrane patches containing £ 2 active channels. The maximum number of simultaneous openings determined the number of active channels during the course of an experiment. To determine the single channel current amplitude, the recorded 120-second data from AF508-CFTR activity was filtered off-line at 100 Hz and then used to construct all the amplitude histogram points that were fitted with functions. muítigausianas using Bio-Patch Analysis software (Bio-Logic Comp. France). Total microscopic current and open probability (Pq) were determined from 120 seconds of channel activity. P_or was determined using Bio-Patch or P ratio software_or = I / i (N), where I = average current, i = single channel current amplitude and N = number of active channels in the patch.

12. Solutions

Extracellular solution (in mM): NMDG (150), aspartic acid (150), CaCl₂ (5), MgCl₂ (2) and HEPES (10) (pH adjusted to 7.35 with Tris base).

Intracellular solution (in mM): NMDG-C1 (150), MgCl₂ (2), EGTA (5), TES (10) and Tris (14) base (pH adjusted to 7.35 with HC1).

13. Cell culture

NIH3T3 mouse fibroblasts stably expressing AF508-CFTR are used for registration of the cut membrane clamp patch. Cells are maintained at 37 ° C at 5% C0₂ and 90% humidity in Dulbecco's modified Eagle's medium supplemented with 2mM glutamine, 10% fetal bovine serum, 1X NEAA, β-ΜΕ, 1X pen / strep and 25mM HEPES in 175cm culture flasks² . For single channel recordings, 2,500-5,000 cells were seeded on poly-L-1 isine coated glass coverslips and cultured for 24-48 hours at 27 ° C before use.

Using the procedures described above, activity has been measured, i.e.

EC₅or, of compound 1 and is shown in Table 4.

Table 4.

<td colspan="3">IC50 / EC50, classifications +++ ^ 2.0 &lt;++ ^ 5.0 &lt;+</td>

<td colspan="3">Percent activity, ratings: + ^ 25.0 &lt;++ ^ 100.0 &lt;+++</td>

<td>Comp. No.</td><td>EC_5th Classified</td><td>Maximum efficiency classified</td>

<td> 1</td><td> ++ +</td><td> ++ +</td>

It is noted that in relation to this date, the best method known by the applicant to put the aforementioned invention into practice is the one that is clear from the present description of the invention.