Pharmaceutical compositions and administrations thereof

[0001] This application claims provisional US priority 61 / 327.095, April 2010. The total priority content is incorporated by reference.

of the application submitted on 22 the application for

FIELD OF THE INVENTION [0002] The present invention relates to pharmaceutical compositions comprising a compound of the formula I in combination with one or both compounds of the formula II and / or compounds of the formula III. The invention also relates to pharmaceutical formulations thereof and methods of using such compositions in the treatment of diseases mediated by

CFTR, in particular, guide fibrosis.

BACKGROUND [0003] Cystic fibrosis (CF) is a recessive genetic disease that affects approximately 30,000 children and adults in the United States and about 30,000 children and adults in

Europe. Despite the progress in the treatment of CF, there is no cure.

[0004] CF is produced by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that encodes an epithelial chloride ion channel responsible for collaborating in the regulation of sai and water absorption and

<img file="AR080944A1_D0001.tif" />

Tr Y&gt;

secretion in various tissues. Small molecule drugs, known as enhancers that increase the probability of opening CFTR channels, represent a potential therapeutic strategy for the treatment of CF. Enhancers of this type are disclosed in WO.

2006/002421, which is incorporated herein by reference in its entirety. Another potential therapeutic strategy includes small molecule drugs known as CF correctors that increase the amount and function of CFTR channels.

Correctors of this type are disclosed in WO.

2005/075435, which are incorporated herein by reference in its entirety.

[0005] Specifically, the CFTR is an anion channel mediated by cAMP / ATP that is expressed in a variety of cell types, including secretory and absorbent epithelial cells, where it regulates the flow of anions through the membrane, as well as the activity of other channels of ions and proteins. In epithelial cells, the normal functioning of the CFTR is of paramount importance for the maintenance of electrolyte transport throughout the body, including respiratory and digestive tissue. The CFTR is composed of about 1,480 amino acids that encode a protein formed by a tandem repeat of transmembrane domains, which contains six transmembrane helices and a nucleotide binding domain.

The two transmembrane domains are linked by a large polar regulatory (R) domain with multiple sites of

<img file="AR080944A1_D0002.tif" />

phosphorylation that regulate the cell.

channel activity and traffic

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[0006] The gene encoding the 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, J .

R. et al. (1989) Science 245: 1066-1073). A defect in this gene causes mutations in the CFTR, which results in cystic fibrosis (CF), the most common terminal genetic disease in humans. Cystic fibrosis affects about one in each

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 defective gene with no apparent effects of the disease. In contrast, people with two copies of the gene associated with CF suffer from the debilitating and terminal effects of CF, including chronic lung disease.

[0007] In patients with CF, CFTR mutations expressed endogenously in respiratory epithelium lead to reduced secretion of apical anions resulting in an imbalance in the transport of ions and fluids. The resulting reduction in anion transport contributes to the accumulation of increased mucus in the lungs and consequent microbial infections that ultimately cause death in patients with CF. In addition to respiratory disease, CF patients typically suffer from gastrointestinal problems and pancreatic insufficiency that, if left untreated, leads to death. In addition, most men

<img file="AR080944A1_D0004.tif" />

with cystic fibrosis they are infertile and fertility is reduced among women with cystic fibrosis. Unlike the serious effects of two copies of the gene associated with CF, people with a single copy of the gene associated with CF show a greater resistance to cholera and dehydration resulting from diarrhea, perhaps which explains the frequency relatively high of the CF gene within the population.

<td>[0008] An analysis of</td><td>sequence</td><td>of the</td><td>CFTR gene from</td><td>chromosomes of</td>

<td>CF has revealed a</td><td>variety</td><td>from</td><td>mutations that</td><td>produce the</td>

<td>disease (Cutting,</td><td>GR et</td><td>to the.</td><td>(1990) Nature</td><td> 346 : 366-369;</td>

Dean, M. et al. (1990) Celi 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, more than 1,000 mutations that cause the disease in the CF gene have been identified (http://www.genet.sickkids.on.ca/cftr/app). The most frequent mutation is an elimination of phenylalanine at position 508 of the amino acid sequence of CFTR, and is commonly known as AF508-CFTR. This mutation occurs in approximately 7% of cystic fibrosis cases and is associated with a serious disease.

[0009] The removal of residue 508 in AF508-CFTR prevents the nascent protein from folding correctly. This results in the inability of the mutant protein to exit

ER, and circulates to the plasma membrane. As a result of elio, the amount of channels present in the membrane is much smaller than that observed in cells expressing a CFTR of type

<img file="AR080944A1_D0005.tif" />

hl w

wild. In addition to difficult traffic, the mutation results in a defective channel opening. All together, the reduced amount of channels in the membrane and the defective opening lead to a reduced transport of anions through the epithelium, which leads to a defective transport of ions and fluids. (Quinton, PM (1990), FASEB J. 4: 2709-2727). However, studies have shown that reduced amounts of AF508-CFTR in the membrane are functional, although it is true that less than wild-type CFTR. (Dalemans et al. (1991), Nature Lond. 354: 526-528; Denning et al., Supra;

Pasyk and Foskett (1995), J. Celi. Biochem 270: 12347-50). In addition to AF508-CFTR, other mutations that cause disease in

CFTRs that give as a traffic result, synthesis and / or opening of defective channels could be regulated in increasing or decreasing manner to alter the secretion of anions and modify the disease progression and / or its severity.

[0010] Although the CFTR transports a variety of molecules in addition to anions, it turns out that this function (the transport of anions) represents an element in an important mechanism of ion and water transport through the epithelium. Other elements include the Na + epithelial channel, the Na + / 2Cl- / K + cotransporter, Na + -K + -ATPase pump and the channels

K + of basolateral membrane and are responsible for taking chloride in the cell.

[ooiij These elements work together to achieve directional transport through the epithelium through its selective expression and location within the cell. The

<img file="AR080944A1_D0006.tif" />

chloride is carried out through activity

ENaC and CFTR present in the apical membrane and the

ATPase and Cl ion channels expressed in Na + -K + pump coordinate 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 Cl channels, and result in a vector transport. The arrangement of the Na + / 2Cl- / K + cotransporter, the Na + -K + -ATPase pump and the K + channels of the basolateral membrane on the basolateral surface and the CFTR on the luminal side coordinate chloride secretion through the CFTR on the luminal side . Since water is probably never actively transported properly, its flow through the epithelium depends on small transepithelial osmotic gradients generated by the volumetric flow of sodium and chloride.

[0012] As discussed previously, the elimination of residue 508 in AF508-CFTR is considered to prevent the nascent protein from folding correctly, resulting in the inability of this mutant protein to exit ER, and that circulates to the plasma membrane. As a result of this, insufficient amounts of the mature protein are present in the plasma membrane, and the transport of chloride within epithelial tissues is significantly reduced. In fact, it has been shown that this defective ER cell phenomenon that processes ABC transporters through ER machinery is

<img file="AR080944A1_D0007.tif" />

the essential basis not only of CF disease but of a wide range of other isolated or inherited diseases.

[0013] It was shown that compounds that are enhancers of the CFTR protein, such as those of formula I and compounds that are correctors of CFTR protein, such as those of formula II or formula III, have utility , independently, in the treatment of diseases modulated by CFTR, as cystic fibrosis.

[0014] According to elio, there is a need for new treatments of CFTR-mediated diseases that include corrective compounds and enhancers of CFTR.

[0015] Particularli, there is a need for combination therapies to treat CFTR-mediated diseases, such as cystic fibrosis, which include corrective compounds and enhancers of CFTR.

[0016] More in particular, there is a need for combination therapies to treat CFTR-mediated diseases, such as cystic fibrosis, including compounds that enhance

CFTR, taies as compounds of the formula I, in combination with CFTR corrective compounds taies as compounds of the formula II and / or of the formula III.

[0017] More particularly, there is a need for combination therapies to treat CFTR-mediated diseases, such as cystic fibrosis, including compounds that enhance

CFTR, taies corno compound 1, in combination with CFTR corrective compounds, taies corno compound 2 and / or compound 3.

<img file="AR080944A1_D0008.tif" />

<img file="AR080944A1_D0009.tif" />

SYNTHESIS OF THE INVENTION [0018] These and other needs are met by means of the present invention which relates to pharmaceutical compositions comprising:

A compound of the formula I

<img file="AR080944A1_D0010.tif" />

Formula I or one of its pharmaceutically acceptable salts, where:

Ring A is selected from:

<img file="AR080944A1_D0011.tif" />

R¹ is -CF₃, -CN or -C ^ CCH₂N (CH3) 2;

R² it's hydrogen, -CH₃, -CF₃, -OH or -CH₂OH;

R³ it's hydrogen, -CH₃, -OCH₃ or -CN;

provided that both R² and R³ are not simultaneously hydrogen;

in combination with one or both of:

A compound of formula II

<img file="AR080944A1_D0012.tif" />

Formula II or one of its pharmaceutically acceptable salts, where:

T is -CH₂-, -CH2CH2-, -CF₂-, -C (CH₃)₂- or -C (0) -;

R¹ 'is H, aliphatic Ci_₆halo CF₃, CHF₂, O (aliphatic C₃-₆) ;

and

R^m or R^u2 is Z^dR₉ where :

Z³ It's a link, CONH, SO₂NH, S0₂N (Ci-è alkyl),

CH2NHSO2, CH₂N (CH₃) S0₂, CH2NHCO, C00, S0₂ or CO; and

R₉ is H, aliphatic Ci_₆ or aryl; me

A compound of formula III

<img file="AR080944A1_D0013.tif" />

Formula III or one of its pharmaceutically acceptable salts, where:

R is H, OH, OCH₃ or two R taken together form -OCH₂O- or OCF₂OR-;

R₄ it is H or alkyl;

R₅ is H or F;

R₆ is H or CN;

R₇ is H, -CH₂CH (OH) CH₂OH, -CH2CH2N⁴ (CH₃) 3 or -CH₂CH₂OH;

&gt; ιιογ „λ î&gt; / ω

Rg is H, OH, -CH₂CH (OH) CH₂OH, -CH₂OH or R₇ and Rg taken together form a five-member ring.

[0019] In another aspect, the pharmaceutical composition comprises compound 1

TO

N

H compound 1 in combination with compound 2 and / or compound 3.

<img file="AR080944A1_D0014.tif" />

<img file="AR080944A1_D0015.tif" />

<img file="AR080944A1_D0016.tif" />

compound 3 [0020] In one aspect, the pharmaceutical composition comprises compound 1, compound 2 and compound 3.

[0021] In another aspect, the invention relates to a pharmaceutical composition comprising at least one column component.

A of Table I and at least one component of column B and / or column C of Table I. These components are described in the corresponding sections of the following pages as embodiments of the invention. For convenience, Table I

<img file="AR080944A1_D0017.tif" />

indicates the section number and the corresponding title of the embodiments of the compounds. For example, the embodiments of the compounds of the formula I are disclosed in section II.Al of this specification.

Table I of Forms of Realization

Forms realization

Column A

Column B

Column C

<td>Section</td><td>Title</td><td>Section</td><td>Title</td><td>Section</td><td>Title</td>

<td>II.Al</td><td>The</td><td>II.Bl</td><td>The</td><td>II.Cl</td><td>The</td>

<td></td><td>compounds</td><td></td><td>compounds</td><td></td><td>compounds</td>

<td></td><td>of the</td><td></td><td>of the</td><td></td><td>of the</td>

<td></td><td>formula I</td><td></td><td>formula II</td><td></td><td>formula</td>

<td></td><td></td><td></td><td></td><td></td><td>III</td>

<td>II.A.2.</td><td>compound</td><td>II.B.2.</td><td>compound</td><td>II.C.2.</td><td>compound</td>

<td></td><td> 1</td><td></td><td> 2</td><td></td><td> 3</td>

[0022] In one aspect, the invention includes a pharmaceutical composition comprising a component selected from any embodiment described in Column A of the

Table I in combination with a component selected from any embodiment described in Column B and / or a component selected from any embodiment described in Column C in Table I.

<img file="AR080944A1_D0018.tif" />

[0023] Thus, in one embodiment, the invention relates to a pharmaceutical composition comprising a compound of formula I and a compound of formula II.

[0024] In another embodiment, the invention relates to a pharmaceutical composition comprising a compound of formula I and a compound of formula III.

[0025] In another embodiment, the invention relates to a pharmaceutical composition comprising a compound of the formula I, a compound of the formula II and a compound of the formula III.

[0026] In another embodiment, the invention relates to a pharmaceutical composition comprising compound 1 and a compound of formula II.

[0027] In another embodiment, the invention relates to a pharmaceutical composition comprising compound 1 and a compound of formula III.

[0028] In another embodiment, the invention relates to a pharmaceutical composition comprising compound 1, a compound of formula II and a compound of formula III.

10029] In another embodiment, the invention relates to a pharmaceutical composition comprising compound 1 and compound 2.

[0030] In another embodiment, the invention relates to a pharmaceutical composition comprising compound 1 and compound 3.

<img file="AR080944A1_D0019.tif" />

bas [0031] In another embodiment, the invention relates to a pharmaceutical composition comprising compound 1, compound 2 and compound 3.

[0032] In another embodiment, the invention relates to a pharmaceutical composition comprising a compound of formula I and compound 2.

[0033] In yet another embodiment, the invention relates to a pharmaceutical composition comprising a compound of the formula I and compound 3.

[0034] In yet another embodiment, the invention relates to a pharmaceutical composition comprising a compound of the formula I, compound 2 and compound 3.

[0035] Various components listed in the

Table I and can be found in US Pat.^Q 7,776,905, US Patent No.^and 7,645,789, US documents 2010/0113508, US

2010/0130547, US 2008 / 0113985A1, US2008 / 0019915A1, US

2008 / 0306062A1, US 2009/0170905 Al, US 2009/0176839 and US

2010/0087490, whose contents are incorporated herein by reference.

DETAILED DESCRIPTION

I. Definitions [0036] Such as is used herein, the following definitions should apply, unless otherwise indicated.

[0037] The term ABC transporter such as used herein implies an ABC transporter protein or one of its

<img file="AR080944A1_D0020.tif" />

fragments comprising at least one binding domain, in doride said protein or fragment is present in vivo or in vitro. The term "binding domain" such as "used herein" implies a domain in the ABC transporter that can be linked with a modulator. See, for example, Hwang, TC et al., J. Gen.

Physiol (1998): 111 (3), 477-90.

[0038] The term CFTR as such is used herein to imply transmembrane cystic fibrosis conductance regulator or one of its mutations capable of regulating activity, including, but not limited to, LF508 CFTR, R117H CFTR and G551D

CFTR (see, for example, http://www.genet.sickkids.on.ca/cftr/, for CFTR mutations).

[0039] Such as is used herein, the term "pharmaceutically active ingredient" or "API" refers to a biologically active compound. Example APIs include the CF enhancer N- (4- (7-azabicyclo [2,2,1] heptan-7-yl) -2 (trifluoromethyl) phenyl) -4-oxo-5- (trifluoromethyl) -1 , 4-dihydroquinolin-3-carboxamide (compound 1). Example APIs also include the 3- (6- (1- (2,2-Difluorobenzo [d] [1,3] dioxol-5-yl) cyclopropancarboxamido) -3-methylpyridin-2-yl) benzoic acid CF correctors (compound 2 ) and (R) -1- (2,2difluorobenzo [d] [1,3] dioxol-5-yl) -N- (1- (2,3-dihydroxypropyl) -6fluoro-2- (l-hydroxy-2 -methylpropan-2-yl) -lH-indole-5yl) cyclopropanecarboxamide (compound 3).

[00401 The term modular such horn used herein implies to increase or reduce with a measurable amount.

<img file="AR080944A1_D0021.tif" />

[0041] The term normal CFTR or a normal CFTR function such as used herein means wild type CFTR without alteration due to environmental factors such as smoking, pollution or anything that causes inflammation in the lungs.

[0042] The term reduced CFTR or reduced CFTR function such as used herein means less than normal CFTR or less than a normal CFTR function.

[0043] As used herein, the term "amorphous" refers to a solid material that does not have a long-range order in the position of its molecules. Amorphous solids, in general, are supercooled liquids where the molecules are randomly arranged so that they do not have a well-defined arrangement, for example, molecular packaging, and no long-range order, amorphous solids, in general, are isotropic, that is, they show similar properties in all directions and do not have defined melting points. For example, an amorphous material is a solid material without characteristic sharp crystalline peaks in its ray power diffraction pattern

X (XRPD) (that is, it is not crystalline as determined by XRPD). Instead, one or several maximum wide peaks (for example, halos) appear in your XRPD pattern. Wide peaks are characteristic of an amorphous solid. Refer to US 2004/0006237 to compare the XRPDs of an amorphous material and a crystalline material.

<img file="AR080944A1_D0022.tif" />

[0044] As used herein, the term "substantially amorphous" refers to a solid material that has little or no long-range order in the position of its molecules. For example, substantially amorphous materials have less than about 15% crystallinity (for example, less than about 10% crystallinity or less than about 5% crystallinity). Also, it should be noted that the expression substantially amorphous includes the descriptor, amorphous, which refers to materials without any (0%) crystallinity.

[0045] As used herein, the term "dispersion" refers to a dispersion system where a substance, the dispersed phase, is distributed in discrete units throughout a second substance (the continuous phase or vehicle). The size of the dispersed phase can vary considerably (for example, single molecules, colloidal particles of nanometer size, up to a size of multiple microns). In general, the dispersed phases can be solid, liquid or gaseous. In the case of a solid dispersion, the dispersed and continuous phases are both solid.

In pharmaceutical applications, a solid dispersion may include: an amorphous drug in an amorphous polymer; an amorphous drug in a crystalline polymer; a crystalline drug in an amorphous polymer; or a crystalline drug in a crystalline polymer. In this invention, a solid dispersion may include an amorphous drug in an amorphous polymer or a drug.

<img file="AR080944A1_D0023.tif" />

amorphous in a crystalline polymer. In some embodiments, a solid dispersion includes the polymer that constitutes the dispersed phase, and the drug constitutes the continuous phase. Or, a solid dispersion includes the drug that constitutes the dispersed phase, and the polymer constitutes the continuous phase.

[0046] As used herein, the term "solid dispersion" refers, in general, to a solid dispersion of two or more components, usually one more drugs (for example, a drug (as is, compound 1)) and a polymer, but possibly containing other components such as surfactants or other pharmaceutical excipients where the drug (s) (for example compound 1) are substantially amorphous (for example, with about 15% or less (for example, about 10% or less, or about 5% or less)) of the crystalline drug (eg empio, N- (4- (7-azabicyclo [2,2,1 ] heptan-7-yl) -2 (trifluoromethyl) phenyl) -4-oxo-5- (trifluoromethyl) -1,4dihydroquinolin-3-carboxamide) or amorphous (i.e., without crystalline drug), and physical stability and / or dissolution and / or solubility of the amorphous or substantially amorphous drug is enhanced by the other components. Solid dispersions typically include a compound dispersed in an appropriate carrier medium, such as a solid state carrier.

For example, a carrier comprises a polymer (for example, a water-soluble polymer or a partially water-soluble polymer) and

<img file="AR080944A1_D0024.tif" />

it may include optional excipients such as functional excipients (for example, one or more surfactants) or non-functional excipients (for example, one or more fillers).

Another illustrative solid dispersion is a joint precipitate or a joint fusion of N- (4- (7-azabicyclo [2,2,1] heptan-7-yl) -2 (trifluoromethyl) phenyl) -4-oxo-5- ( trifluoromethyl) -1,4 dihydroquinolin-3-carboxamide with at least one polymer.

[0047] A joint precipitate is a product after the dissolution of a drug and a polymer in a solvent or in a solvent mixture followed by the removal of the solvent or solvent mixture. Sometimes, the polymer can be suspended in the solvent or in the solvent mixture. The solvent or solvent mixture includes organic solvents and super critical fluids. A joint fusion is a product after the heating of a drug and a polymer so that they are optionally joined in the presence of a solvent or a mixture of solvent, followed by mixing, the removal of at least a portion of the solvent, if applicable , and cooling at room temperature at a selected speed.

[0048] Such a horn is used herein, crystalline refers to compounds or compositions wherein the structural units are arranged in fixed geometric patterns or networks, so that the crystalline solids have a rigid long range order. The structural units that constitute the crystalline structure can be atoms, molecules or ions. The crystalline solids show defined melting points.

<img file="AR080944A1_D0025.tif" />

[0049] Such a horn is used herein, the phrase "substantially crystalline" means a solid material that is arranged in fixed geometric patterns or networks that have a rigid long range order. For example, substantially crystalline materials have more than about 85% crystallinity (for example, more than about 90% crystallinity or more than about 95% crystallinity).

It is also noted that the expression "substantially crystalline" includes the "crystalline descriptor"¹, which is defined in the previous paragraph.

[0050] As used herein, the term "crystallinity" refers to the degree of structural order in a solid. For example, compound 1, which is substantially amorphous, has less than about 15% crystallinity, or its solid state structure is less than about

15% crystalline In another example, compound 1, which is amorphous, has a crystallinity of zero (0%).

[0051] As used herein, the term "excipient" is an inactive principle in a pharmaceutical composition. Examples of excipients include fillers or diluents, surfactants, binders, glidants, lubricants, disintegrants and the like.

[0052] As used herein, a disintegrant is an excipient that hydrates a pharmaceutical composition and assists in the dispersion of the tablet. Examples of disintegrants include sodium croscarmellose and / or sodium starch giocolate.

[0053] As used herein, the term "diluent" or "filled" expression is an excipient that brings volume to a pharmaceutical composition. Examples of fillers include: lactose, sorbitol, celluloses, calcium phosphates, starches, sugars (for example, mannitol, sucrose or the like) or any combination thereof.

[0054] As used herein, the term "surfactant" is an excipient that imparts pharmaceutical compositions with improved solubility and / or wettability. Examples of surfactants include sodium lauryl sulfate (SLS), sodium stearyl fumarate (SSF), polyoxyethylene sorbitan mono-oleate (for example Tween ™), or any other combination thereof.

10055] As used herein, the term "binder" is an excipient that imparts a pharmaceutical composition with improved cohesion or improved tensile strength (for example, hardness). Examples of binders include dibasic calcium phosphate, sucrose, corn starch, microcris talin cellulose and modified cellulose (eg, hydroxymethyl cellulose).

<td>[0056] Of the</td><td>form used in</td><td>the</td><td>I presented,</td><td>the term</td>

<td>slider</td><td>it's an excipient</td><td>what</td><td>teaches</td><td>compositions</td>

<td>pharmaceuticals</td><td>with properties of</td><td>f lu jo</td><td>best prayers</td><td>Between the</td>

Examples of sliders include colloidal silica and / or talc.

<img file="AR080944A1_D0026.tif" />

[0057] As used herein, the term "dye" is an excipient that imparts a pharmaceutical composition with a convenient color. Examples of dyes include commercially available pigments taies corno azul N.^s 1 Aluminum Lake by FD&amp;C, blue N. ⁹ 2 of FD&amp;C, other blue colors of FD&amp;C, titanium dioxide, iron oxide and / or combinations thereof.

[0058] As used herein, a lubricant is an excipient that is added to pharmaceutical compositions that are pressed to form tablets. The lubricant collaborates in the compaction of granules to form tablets and in the ejection of a tablet of a pharmaceutical composition of the press matrix. Examples of lubricants include magnesium stearate, stearic acid (stearin), hydrogenated oil, sodium stearyl fumarate or any combination thereof.

[0059] As used herein, the term "friability" refers to the property that a tablet has of remaining intact and maintaining its shape despite an external pressure force. Friability can be measured by using the mathematical expression presented in the

Equation 1:

% friction W00x (Υ, -Υ) where W_or is the initial weight of the tablet and W_t It is the final weight of the tablet after being passed through the friabilizer.

(1

<img file="AR080944A1_D0027.tif" />

(0060] Friability is measured by the use of a standard USP test apparatus that rotates experimental tablets at 100 revolutions. Some tablets of the present invention have a friability of less than about 1% (for example, less than about the

0.75%, less than about 0.50%, or less than about 0.30%).

[0061] As used herein, the term "average particle diameter" is an average particle diameter of the shape measured using techniques such as laser dispersion, image analysis or sieve analysis.

[0062] As used herein, the term "volume density" is the mass of particles of material divided by the total volume occupied by the particles. The total volume includes the particle volume, the empty volume between particles and the internal pore volume. Volumetric density is not an intrinsic property of a material; You can change the way you process the material.

[0063] The term aliphatic or aliphatic group as used herein means a linear (ie, unbranched) or branched chain, substituted or unsubstituted hydrocarbon chain that is completely saturated or that contains one or more units of unsaturation or a monocyclic hydrocarbon or a bicyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic (also mentioned herein

<img file="AR080944A1_D0028.tif" />

cycloaliphatic carbocycle or cycloalkyl), which has a unique junction point with the rest of the molecule. Unless otherwise specified, aliphatic groups contain

1-20 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 C-Cg monocyclic hydrocarbon or a hydrocarbon

C₈-Ci4 bicyclic or tricyclic that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic, which has a unique point of union with the rest of the molecule, where any individual ring in said bicyclic ring system has 3-7 members Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl, and their taies corno (cycloalkyl) alkyl, (cycloalkenyl) alkyl or (cycloalkyl) alkenyl hybrids. Appropriate cycloaliphatic groups include cycloalkyl, bicyclic cycloalkyl (eg, decalin), bicycloalkyl in taies corno norbornyl bridge or [2,2,2] bicyclo-octyl or tricyclic in taies corno adamantyl bridge.

<img file="AR080944A1_D0029.tif" />

10064] The term "heteroaliphatic", as used herein, implies aliphatic groups wherein one or two carbon atoms are independently replaced by one or more of oxygen, sulfur, nitrogen, phosphorus or silicon. Heteroaliphatic groups may be substituted or unsubstituted, branched or unbranched, cyclic or acyclic and include heterocycle, heterocyclyl, heterocycloaliphatic or heterocyclic groups.

[0065] The term "heterocycle, heterocyclyl, heterocycloaliphatic or heterocyclic" as used herein implies 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 members of the ring in which one or more members of the ring is a heteroatom selected, independently, from oxygen, sulfur, nitrogen or phosphorus and each ring In the system it contains 3 to 7 ring members.

[0066] The term "heteroatom" implies 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 (horn in 3,4-dihydro-2H-pyrrolyl), NH (horn in pyrrolidinyl) or NR '(horn in pyrrolidinyl substituted with N)).

<img file="AR080944A1_D0030.tif" />

[0067] The unsaturated expression, as used herein, means that a remainder has one or more units of unsaturation.

[0068] 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 having a total of 5 to 14 ring members, where the At least one ring in the system is aromatic and where 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 such as is defined herein below.

[0069] An aliphatic or heteroaliphatic group or a non-aromatic heterocyclic ring may contain one or more substituents. Appropriate substituents on saturated carbon of an aliphatic or heteroaliphatic group or of a non-aromatic heterocyclic ring are selected from those listed above for unsaturated carbon of an aryl or heteroaryl group and additionally include the following: = 0, = S, = NNHR *, = NN (R'b, = NNHC (O) R *, = NNHCO₂(alkyl), = NNHSO₂ (alkyl) o = NR *, where each R * is independently selected from hydrogen or a cyclic aliphatic₆ optionally substituted. Optional substituents in the aliphatic group of R * are selected from NH₂, NH (aliphatic Cm), N (aliphatic Ct-₄)₂, halo, aliphatic Ci-4, OH, O (aliphatic C1-4), NO₂, CN, CO₂H, CO₂ (aliphatic

<img file="AR080944A1_D0031.tif" />

Ci-₄), 0 (C1-4 haloaliphatic) ο halo (Cm aliphatic), where each

<img file="AR080944A1_D0032.tif" />

one of the aliphatic groups Ci_₄ R's previous is not substituted.

[0070] The substituents optionally in the nitrogen of a non-aromatic heterocyclic ring are selected from -R⁴, N (R⁺) 2, -C (O) R⁺, -CO2R⁺, -C (O) C (O) R⁺, -C (O) CH2C (O) R⁴, -so2r⁴,

SW₂N (R ')₂, -C (= S) N (R *) 2, -C (= NH) -N (R ') 2 O -NR'SO₂R '; wherein R 'is hydrogen, an optionally substituted C1-6 aliphatic, optionally substituted phenyl, -O (Ph) optionally substituted,

CH₂(Ph) optionally substituted, - (CH₂) i-₂(Ph) optionally substituted; -CH = CH (Ph) optionally substituted; or an unsubstituted 5-6 membered heteroaryl or heterocyclic ring having one to four heteroatoms independently selected from oxygen, nitrogen or sulfur, or, despite the above definition, two independent occurrences of R⁺, in the same substituent or in different substituents, taken together with the or the atoms to which each R * group is attached, form a 3 to 8 membered cycloalkyl ring, heterocyclyl, aryl or heteroaryl with 0-3 heteroatoms selected, of independent mode of nitrogen, oxygen or sulfur. Optional substituents in the aliphatic group or the phenyl ring of R * are selected from NH₂, NH (aliphatic Cm), N (aliphatic CmL ·, halo, aliphatic Ci-4, OH, O (aliphatic C4-4), NO₂, CN, CO₂.H, CO₂(aliphatic

C1-4), O (C1-4 aliphatic halo) or halo (Cm aliphatic Cm), where each of the Ci_ aliphatic groups₄ previous of R⁺ It is not substituted.

<img file="AR080944A1_D0033.tif" />

SVÛ '[0071] As detailed above, in some embodiments, two independent occurrences of R (or any other variable similarly defined herein), are taken together with the one or all the atoms to which each variable is joined to form a 3-8 membered cycloalkyl ring, heterocyclyl, aryl or heteroaryl with 0-3 heteroatoms independently selected from nitrogen, oxygen or sulfur. Example rings that are formed when two independent occurrences of r '(or any other variable that is defined similarly herein) are taken together with the or the atoms to which each variable is attached include, but is not limited to, the following: a) two independent occurrences of r '(or any other variable similarly defined herein) that are attached to the same atom and are 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-l-yl, piperazin-l-yl or morpholin-4il group; and b) two independent occurrences of r '(or any other variable similarly defined herein) that bind to different atoms and are taken together with the two of these atoms to form a ring, for example, where a phenyl group is rr °^R0 replaced with two occurrences of OR v, these two occurrences of R ° are taken together with the oxygen atoms to which they are attached to form a fused ring containing

<img file="AR080944A1_D0034.tif" />

<img file="AR080944A1_D0035.tif" />

6-member oxygen: 4 θ. It will be appreciated that a variety of other rings may be formed when two independent occurrences of r '(or any other variable defined in a similar manner herein) are taken together with the or the atoms to which each variable is attached and the detailed examples previously they are not intended to be limiting.

[0072] A substituent bond, for example, in a bicyclic ring system, such as indicated below, means that the substituent can be attached to any atom of the substitutable ring in one of the rings of the bicyclic ring system:

(WR^w)_m

10073] The term "protecting group (PG)" as used herein, represents those groups that are intended to protect a functional group, such as, for example, an alcohol, amine, carboxyl, carbonyl, etc., against undesirable reactions during Synthesis procedures Commonly used protective groups are described in Greene and Wuts, Protective

Groups in Organic Synthesis, 3^rd Edition (John Wiley &amp; Sons, New

York, 1999), which is incorporated herein by reference. Examples of nitrogen protecting groups include acyl, aroyl or carbamyl groups such as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl, phthalyl, or 29

<img file="AR080944A1_D0036.tif" />

chlorobenzyloxycarbonyl, nitrobenzyloxycarbonyl, nitrophenoxyacetyl, α-chlorobutyryl, benzoyl, 4-chlorobenzoyl,

4-bromobenzoyl, 4-nitrobenzoyl and chiral auxiliaries taies as protected or unprotected amino acids D, L or D, L taies as alanine, leucine, phenylalanine and the like; sulfonyl groups taies as benzenesulfonyl, p-toluenesulfonyl and the like; carbamate taies groups such as benzyloxycarbonyl, pp-methoxybenzyloxycarbonyl, p2-nitrobenzyloxycarbonyl, pbromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl, 3,5-dimethoxybenzyloxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyloxycarbonyloxycarbonyloxycarbonyloxycarbonyloxycarbonyloxycarbonyloxycarbonyloxycarbonyloxycarbonyloxycarbonyloxycarbonyloxycarbonyloxycarbonyloxycarbonyloxycarbonyloxycarbonyloxycarbonyloxycarbonyloxycarbonyloxycarbonyloxycarbonyloxycarbonyloxycarbonyloxycarbonyloxycarbonyl

1- (p-Biphenyl 1) -1-methylethoxycarbonyl, a, a-dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxycarbonyl, t-butoxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, methoxycarbonyl, 2,2-ethoxycarbonyloxycarbonyloxycarbonyloxycarbonyloxycarbonyloxycarbonyloxycarbonyloxycarbonyloxycarbonyloxycarbonyl, phenoxycarbonyl, 2,2-ethoxycarbonyl, phenoxycarbonyloxycarbonyloxycarbonyl, phenoxycarbonyl, 2,2-ethoxycarbonyl, phenoxycarbonyloxycarbonyloxycarbonyl, phenoxycarbonyl, phenoxycarbonyloxycarbonyl, phenoxycarbonyl, phenoxycarbonyloxycarbonyl, phenoxycarbonyloxycarbonyl nitrophenoxycarbonyl, fluorenyl-9methoxycarbonyl, cyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl and the like, arylalkyl groups taies as benzyl, triphenylmethyl, benzyloxymethyl and the like and silyl groups taies as trimethylsilyl and the like. Preferred N protecting groups are tert-butoxycarbonyl (Boc).

[0074] Examples of useful protecting groups for acids are alkyl esters substituted taies corno

<img file="AR080944A1_D0037.tif" />

fluorenylmethyl, methoxymethyl, tetrahydropyranyl metiltiorfo, tetrahydrofuranyl, methoxyethoxymethyl, 2- (trimethylsilyl) ethoxymethyl, benzyloxymethyl, pivaloyloxymethyl, fenilacetoximetilo, triisopropropilsisililmetilo, cyanomethyl, acetol, phenacyl, substituted phenacyl esters, 2,2,2- trichloroethyl, 2-haloethyl, ω-chloroalkyl , 2 (trimethylsilyl) ethyl, 2-methylthioethyl, t-butyl, 3-methyl-3pentyl, dicyclopropylmethyl, cyclopentyl, cyclohexyl, allyl, metalyl, cinnamonyl, phenyl, silyl esters, benzyl and bendi substituted esters, 2,6-dialkylphenyl esters such as pentafluorophenyl, 2,6-dialkylpihenyl. Preferred protecting groups for acids are methyl or ethyl esters.

[0075] The methods of addition (a process mentioned in general as protection) and elimination (process mentioned in general as deprotection) such as amine and acid protecting groups are well known in the art and are available, for example, in PJ Kociensky , Protecting Groups, Thieme, 1994, which is incorporated herein by reference in its entirety and in Greene and Wuts, Protective Groups in Organic Synthesis, 3^rd

Edition (John Wiley &amp; Sons, New York, 1999).

[0076] If otherwise stated, the structures represented herein 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, isomers (Z) and (E) double bond e

<img file="AR080944A1_D0038.tif" />

conformational isomers (Z) and (E). Accordingly, individual stereochemical isomers as well as the enantiomeric, diastereomeric and geometric (or conformational) mixtures of the present compounds are within the scope of the invention. Unless stated otherwise, all tautomeric forms of the compounds of the invention are within the scope of the invention. For example, the compounds of the formula I may exist as tautomers:

<img file="AR080944A1_D0039.tif" />

[0077] Additionally, unless otherwise stated, the structures represented herein also include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, the compounds having the present structures except in the replacement of hydrogen by deuterium or tritium, or the replacement of a carbon by a carbonⁱ³C- o ^{one z}C-enriched are within the scope of the invention. Such compounds are useful, for example, as analysis tools or probes in biological assays.

10078] Examples of suitable solvents are, without limitation, water, methanol, dichloromethane (DCM), acetonitrile, dimethylformamide (DMF), ethyl acetate (EtOAc), isopropyl alcohol (IPA), isopropyl acetate (IPAc),

<img file="AR080944A1_D0040.tif" />

tetrahydrofuran (THF) methylpyrrolidone (NMP).

methyl ethyl ketone (MEK),

<img file="AR080944A1_D0041.tif" />

II. THE COMPOUNDS OF THE INVENTION [00791 In one aspect, the invention relates to a pharmaceutical composition comprising a compound of formula I in combination with a compound of formula II and / or a compound of formula III.

<img file="AR080944A1_D0042.tif" />

Formula I

<img file="AR080944A1_D0043.tif" />

<img file="AR080944A1_D0044.tif" />

<img file="AR080944A1_D0045.tif" />

Formula II

<img file="AR080944A1_D0046.tif" />

Formula IT T

II.A. The compounds of formula I

<img file="AR080944A1_D0047.tif" />

<img file="AR080944A1_D0048.tif" />

<img file="AR080944A1_D0049.tif" />

Formula I

II.A.1. Embodiments of the compounds of the formula I [0080] In one aspect, the compositions of the invention include a compound of the formula I

<img file="AR080944A1_D0050.tif" />

Formula I or one of its pharmaceutically acceptable salts, where:

Ring A is selected from:

_nì i ri Ti^H^Hx OH

<img file="AR080944A1_D0051.tif" />

<img file="AR080944A1_D0052.tif" />

<img file="AR080944A1_D0053.tif" />

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

<td>R¹ is</td><td>-CF₃, -CN 0</td><td>-C =</td><td>CCH₂N (CH₃) ₂;</td>

<td>R² is</td><td>hydrogen,</td><td>-ch₃</td><td>, -CF₃, -OH ο -CH2OH;</td>

<td>R³ is</td><td>hydrogen,</td><td>-ch₃</td><td>, -OCH₃ 0 -CN;</td>

<td>always</td><td>that both of them</td><td>R²</td><td>and R³ do not be simultaneously</td>

hydrogen;

[0081] In one embodiment, ring A of the formula I

-N

<img file="AR080944A1_D0054.tif" />

is

<img file="AR080944A1_D0055.tif" />

<img file="AR080944A1_D0056.tif" />

[0082] In one embodiment, ring A of the formula I is (b).

[0083] In another embodiment, ring A of formula I ^HOxH

<img file="AR080944A1_D0057.tif" />

en [0084] In yet another embodiment, the A ring of the .OH

<img file="AR080944A1_D0058.tif" />

Formula I is (d).

<img file="AR080944A1_D0059.tif" />

<td> [0085]</td><td>In</td><td>a</td><td>embodiment,</td><td>R¹</td><td>of formula I is -CF₃.</td>

<td> [0086]</td><td>In</td><td>other</td><td>embodiment,</td><td>r '</td><td>¹ of the formula I is -CN.</td>

<td> [0087]</td><td>In</td><td>other</td><td colspan="2">. embodiment,</td><td>R¹ of the formula I is -C = -</td>

<td>CCH₂Neither</td><td>(CH₃ !</td><td>OR. ·</td><td></td><td></td><td></td>

<td> [0088]</td><td>In</td><td>a</td><td>embodiment,</td><td>r²</td><td>of the formula I is -CH₃.</td>

<td> [0089]</td><td>In</td><td>other</td><td>embodiment,</td><td>R '</td><td>² of formula I is -CF₃.</td>

<td> [0090]</td><td>In</td><td>other</td><td>embodiment,</td><td>R '</td><td>² of the formula I is -OH.</td>

<td> [0091]</td><td>In</td><td>other</td><td>embodiment,</td><td>R '</td><td>² of the formula I is -CH₂OH</td>

<td> [0092]</td><td>In</td><td>a</td><td>embodiment,</td><td>R³</td><td>of the formula I is -CH₃.</td>

<td> [0093]</td><td>In</td><td>a</td><td>embodiment,</td><td>R³</td><td>of formula I is -OCH₃.</td>

<td> [0094]</td><td>In</td><td>other</td><td>embodiment,</td><td>R^:</td><td>³ of the formula I is -CN.</td>

10095] In one embodiment, hydrogen; and R³ of the formula I is -CH₃, [0096] In another embodiment, R²

CF₃, -OH or -CH₂OH; and R³ of the formula I [0097] In various embodiments

<img file="AR080944A1_D0060.tif" />

-OCH₃ or -CN.

of the formula I is -CH₃, is hydrogen.

of the present invention,

Saw ?

Ring A of formula I is, R is -CF₃, R is hydrogen; and R³ is -CH3, -OCH3 or -CN. In other embodiments, R¹ It's -CN In other embodiments, R¹ is C = CCH2N (CH₃ ) 2. In one embodiment, R³ is -CH3. Or R³is -OCH3. Or R³ It's -CN

[0098] In other embodiments of the present invention,

<img file="AR080944A1_D0061.tif" />

ring A of formula I

CF₃, -OH or -CH₂OH and R³realization, R¹ It's -CN In

C ^ CCH₂N (CH₃ ) 2. In one form it is -CF₃. Or R² is -OH

10099] In various forms of is hydrogen. In other forms of other embodiments, R ~ is embodiment, R² is -CH3. Or R²Or R² is -CH2OH.

embodiment of the present invention, the hydrogen ring A; and realization, of the formula I is

R³ is -CH₃, -OCH₃R¹ It's -CN In other f •^N or -CN.

forms of

R¹ is -CF₃, R² is

In other embodiments, R 'is 36

<img file="AR080944A1_D0062.tif" />

C = CCH₂N (CH3)₂ ©

In one embodiment, R³ is -OCH3. 0 biVrif,

R³ is -CH3. Or R³ It's -CN

In other embodiments of the present [00100] .N

<img file="AR080944A1_D0063.tif" />

invention, ring A is, R¹ of formula I is -CF₃, R² is -CH₃, -CF₃, -OH or -CH₂OH and R³ It is hydrogen. In other embodiments, R¹ It's -CN In other embodiments, R^J is C = CCH2N (CH3) 2. In one embodiment, R² is -CH3. Or R²is -CF3. Or R² is -OH Or R² is -CH₂OH

[00101] In various embodiments of the present HO. ,_H

<img file="AR080944A1_D0064.tif" />

invention, ring A of formula I is .N, R 'is -CF₃,

R² it is hydrogen; and R³ is -CH3, -OCH3 or -CN. In other embodiments, R¹ It's -CN In other embodiments, R 'is C ^ CCH2N (CH₃ ) ₂. In one embodiment, R³ is -CH₃. Or R³is -OCH3. Or R³ It's -CN

100102] In other embodiments of the present

HO. .h

<img file="AR080944A1_D0065.tif" />

invention, ring A of formula I is, R is -CF₃,

R² is -CH₃, -CF₃, -OH or -CH₂OH and R³ It is hydrogen. In other embodiments, R¹ It's -CN In other embodiments, R¹ is -C-'CCH2N (CH;) 2. In one embodiment, R² is -CH3. O well,

R² is -CF3. Or R² is -OH Or R² is -CH2OH.

<img file="AR080944A1_D0066.tif" />

[00103] In various embodiments of the dam ^H- OH

<img file="AR080944A1_D0067.tif" />

invention, ring A of formula I is, R¹ is -CF₃,

R² it is hydrogen; and R³ is -CH3, -OCH3 or -CN. In other embodiments, R¹ It's -CN In other embodiments, R¹ is C ^ CCH2N (CH₃) 2 · In one embodiment, R³ is -CH3. Or R³is -OCH3. Or R³ It's -CN

[00104] In other embodiments of the present ^H- .OH

<img file="AR080944A1_D0068.tif" />

Invention, ring A of formula I is, R 'is -CF₃,

R² is -CH3, -CF3, -OH or -CH2OH and R³ It is hydrogen. In other embodiments, R¹ It's -CN In other embodiments, R is -CsCCH2N (CH₃ ) 2. In one embodiment, R² is -CH3. Or R² is -CF3. Or R² is -OH 0 good, R² is -CH₂OH

[00105] Representative compounds of formula I are set forth in the following Table 1-1.

Table 1-1

1-2

<img file="AR080944A1_D0069.tif" />

<img file="AR080944A1_D0070.tif" />

1-3

<img file="AR080944A1_D0071.tif" />

1-4

1-5

1-6

<img file="AR080944A1_D0072.tif" />

1-13 1-14

<img file="AR080944A1_D0073.tif" />

<img file="AR080944A1_D0074.tif" />

II.A.2. Compound 1 [00106] In another embodiment, the compound of formula I is compound 1, which is known by its chemical name

N- (4- (7-Azabicyclo [2,2,1] heptan-7-yl) -2- (trifluoromethyl) phenyl) 4-OXO-5- (trifluoromethyl) -1,4-dihydroquinolin-3-carboxamide.

/ FOLIO) %

<img file="AR080944A1_D0075.tif" />

compound 1

II.A.3

Synthesis of the compounds of formula I

II.A.3.a. GENERAL SCHEMES

Scheme 1-1: Preparation of the compounds of formula I

<img file="AR080944A1_D0076.tif" />

b 1 bc O 2 dd I ι 3. c

<img file="AR080944A1_D0077.tif" />

a) (CO₂R) ₂CH = CH (OR), toluene, heat; b) Dopherm or diphenyl ether, reflux, N2 atmosphere; c) elimination of the halogen blocking group if present (for example, -Cl), Pd / C,

H₂, EtOH; d) removal of the protective group R by base or acid;

e) CH₃CN, Et₃N, heat; f) Pd / C, H₂, EtOH; g) HATU, Et₃N, DMF or

<img file="AR080944A1_D0078.tif" />

Propylphosphonic acid cyclic anhydride (T3P®), pyridine, methyltetrahydrofuran.

100107] Scheme 1-1 represents a convergent approach to the preparation of compounds of formula I from substituted derivatives of benzene la and 2a. In the last transformation, the formation of amide by coupling of carboxylic acid ld with amine 2c to give a compound of the formula

I can be performed using 0- (7azabenzotriazol-l-yl) -N, N, N ', N'-tetramethyluronium (HATU) and triethylamine in N, N-dimethylformamide (DMF) or cyclic anhydride of propylsulfonic acid (T3P) hexafluorophosphate ®) and pyridine in 2-methyltetrahydrofuran. The carboxylic acid ld is prepared from the corresponding substituted benzene derivative by means of a sequence that begins with heat-mediated condensation of it with an appropriate maionate (CO₂R)? CH = CH (OR), where

R is an alkyl or aryl group such as methyl, ethyl, butyl, phenyl, p-nitro phenyl or the like, to provide lb.

<td> 100108]</td><td colspan="2">The compound lb</td><td colspan="2">becomes</td><td>in acid</td><td>carboxylic</td><td>ld</td>

<td>through</td><td>from</td><td colspan="2">a sequence of</td><td>three</td><td>stages</td><td>including</td><td>the</td>

<td>cyclization</td><td colspan="2">intramolecular</td><td>then</td><td>from</td><td>to warm</td><td>at reflux</td><td>in</td>

<td>Dopherm ο</td><td>ether</td><td>say phenyl</td><td>(stage b)</td><td colspan="2">, followed by</td><td>elimination</td><td>(from</td>

<td colspan="2">be necessary)</td><td colspan="2">from the halo group</td><td>from</td><td>blocking</td><td>(stage c)</td><td>in</td>

Palladium catalyzed dehalogenation conditions and acid or base catalyzed saponification (step d). The order of the deprotection and saponification stages can be cjyÎQLIO¹

<img file="AR080944A1_D0079.tif" />

⁴¹ ΙΆ invest; that is, stage c can occur before or after * stage d, as indicated in Scheme 1-1.

[00109] Referring again to Scheme 1-1, aniline 2c derivatives can be prepared from nitrobenzene.

2nd through a sequence of three stages. Thus, the

HN 'coupling of nitrobenzene 2a with a cyclic amine 3 such a horn defined herein in the presence of triethylamine provides compound 2b. Palladium catalyzed reduction of 2b provides amine 2c.

Scheme 1-2: Preparation of the compounds of the formula I.

or₂n

<img file="AR080944A1_D0080.tif" />

OR₂N

Br 4 b, c

<img file="AR080944A1_D0081.tif" />

<img file="AR080944A1_D0082.tif" />

<img file="AR080944A1_D0083.tif" />

DMSO, K₂CO₃, 8 0 ^SC; b)

N, N-dimethylprop-2-in-l-amine,

Pd (PPhj) ₂C1₂, Cui, DMF, TEA, 80 ^QC; c) Faith, FeSO / ,, H₂O or Zn, AcOH,&gt;

ω

<img file="AR080944A1_D0084.tif" />

H₂0; d) HATU, Et₃N, DMF or cyclic propylphosphonic acid anhydride (T3P®), pyridine, 2-methyltetrahydrofuran.

[00110] Scheme 1-2 represents the synthesis of the compounds of formula I which carry a propylamine side chain. In this way, the nitrobenzene coupling

2a, wherein Hai is bromide, chloride or the like, with 3 such horn defined herein in the presence of potassium carbonate in DMSO provides intermediate 4. Coupling of intermediate 4 catalyzed with palladium with N, N-dimethylprop-2- in1-amine, followed by iron or zinc catalyzed reduction of the nitro moiety, provides amine 5. Coupling of amine 5 with carboxylic acid ld provides 6.

<img file="AR080944A1_D0085.tif" />

Scheme 1-3: Preparation of some compounds of the formula I ο, ν

<img file="AR080944A1_D0086.tif" />

R (H or OH) 7

Hai RN

2nd

<img file="AR080944A1_D0087.tif" />

b (R = OH) (H or OH) fa

<img file="AR080944A1_D0088.tif" />

a) DMSO, K₂CO₃, heat or CH₃CN, ASD, heat; b) PGX taies horn

TBDMSC1, taies base as imidazole, DMF; c) H ?, Pd / C, EtOH; d)

HATU, Et₃N, DMF or cyclic anhydride of propic acid 1phosphonic acid (T3P®), pyridine, 2-methyltetrahydrofuran; e) check out of

PG, taies as HCl, EtOH. PG = protective group; X = outgoing group.

100111] Scheme 1-3 represents the synthesis of a compound is 7-azabicyclo [2,2,1] heptane, of the formula I wherein t

<img file="AR080944A1_D0089.tif" />

<img file="AR080944A1_D0090.tif" />

which optionally carries an exo or endohydroxy group at position 2. Adducts substituted with hydroxy (+) - endo-7azabicyclo [2,2,1] heptan-2-ol, (-) - endo-l azabicyclo [2, 2,1] heptan-2-ol, (+) -exo-7-azabicyclo [2,2,1] heptan2-ol and (-) - exo-7-azabicyclo [2,2,1] heptan-2- ol can be prepared using the procedures as described in Fletcher,

SR, et al., Total Synthesis and Determination of the Absolute

Configuration of Epibatidyne, J. Org. Chem, 59, pp. 1771-1778 (1994). 7-azabicyclo [2,2, l] heptane itself is available at Tiger Scientific Inc. stores 324 Stokes Avenue Ewing, NJ,

08638, United States.

100112] Thus, as with the series of transformations outlined in Schemes 1-1 and 1-2, the coupling of compound 2a with the [2,2,1] amine bicycles of formula 7 provides a compound of the formula 8. If the compound of the formula 8 has a hydroxy group, it may be necessary to protect the hydroxy group with a protective group, such as a silyl protecting group as in step b, before further transformations. Treatment of the hydroxylated compound of the formula 8 with a silylating agent such as terbutyldimethylsilyl chloride, using known conditions, provides the protected compound of the formula 9. Reduction of the nitro moiety provides an amine of the formula 10, The formation of amides with ld (comp. Scheme 1-3) and removal of the hydroxy protecting group (step e - if necessary) provides a

<img file="AR080944A1_D0091.tif" />

compound of formula 11 which is also a compound of 1 formula I.

II.A.4. Examples: Synthesis of compound 1 [00113] Intermediary 1: 4-oxo-5- (trifluoromethyl) -1,4 dihydroquinolin-3-carboxylic acid (17).

CF;

TO

Etc

JL toluene '0 E t CF,

TO'

TO,

CF; 0 oc wt he rm E t

0 '' OEt

Cl

NA OEt 25crech Τ 'NHLH

10% Pd / C.

H Et ;. N EtOH, E = C; SC%

CF -, 0 0

<img file="AR080944A1_D0092.tif" />

OEt EM NaOH

EC -C [00114]

Emp emp

CF, 0 0

TO

OEt

<img file="AR080944A1_D0093.tif" />

the :

0H

2- ((2-Chloro-5 (trifluoromethyl) phenylamino) methylene) diethyl malonate (14).

2-Chloro-5- (tri f luoromethyl) aniline 12 (200 g, 1,023 mol), 2 (ethoxymethylene) diethyl mathinate 13 (276 g, 1.3 mol) and toluene (100 mL) were combined under an atmosphere of nitrogen in a 1 liter round base vessel with three mouths equipped with a Dean-Stark condenser. The solution was heated with stirring to 140 ° C and the temperature was maintained for 4 h. The reaction mixture was cooled to 70^QC and hexane (600 mL) was added slowly. The resulting suspension was stirred and allowed to warm to room temperature. The solid was collected by filtration, washed with 10% ethyl acetate in hexane (2x 400 mL) and then dried in vacuo to provide a white solid (3.50 g, 94% yield) as a condensation product.

Desired SvO 2 - ((2-Cl Gold -5 --νΛ / (Trifluoromethyl) Phenylamino) Methylene) Diethyl Masionate 14. 'H NMR (400 MHz, DMSO-ds) δ 11.28 (d, J = 13, 0 Hz, IH), 8.63 (d, J =

13.0 Hz, IH), 8.10 (s, IH), 7.80 (d, J = 8.3 Hz, IH), 7.50 (dd,

J = 1.5, 8.4 Hz, IH), 4.24 (q, J = 7.1 Hz, 2H), 4.17 (q, J = 7.1

Hz, 2 Η), 1.27 (m, 6H).

[00115] Example lb: ethyl 8-chloro-4-oxo-5- (trifluoromethyl) -1,4-dihydroquinolin-3-carboxylate (15).

Method 1 [00116] A 1-liter 3-mouth vessel was charged with

Dopherm® (200 mL, 8 mL / g), which was degassed at 200 -C for 1

h. The solvent was heated to 260^SC and portioned for 10 min with 2 - ((2-chloro-5 (trifluoromethyl) phenylamino) methylene) diethyl 14 (25 g,

0.07 moi). The resulting mixture was stirred at 260 ° C for 6.5 hours (h) and the resulting ethanol byproduct was distilled off. The mixture was allowed to cool slowly 80^SC. Hexane (150 mL) was added slowly for 30 minutes (min), followed by an additional 200 mL of hexane added in one portion. The suspension was stirred until it reached room temperature.

The solid was filtered, washed with hexane (3 χ 150 mL) and then dried in vacuo to provide ethyl 8-chloro-4-oxo-5 (trifluoromethyl) -1,4-dihydroquinolin-3-carboxylate 15 as of a roasted solid (13.9 g, 65% yield). ri

NMR (400 MHz, DMSO-d_s) δ 11.91 (s, IH), 8.39 (s, IH), 8.06 (d, J

<img file="AR080944A1_D0094.tif" />

= 8.3 Hz, IH), 7.81 (d, J = 8.4 Hz, 1H), 4.24 (q, J = 7.1 Hz

2H), 1.29 (t, J = 7.1 Hz, 3H).

Method 2 [00117] Compound 14 (2000 g, 5.468 mol) was introduced into the reactor. Dopherm (4,000 L) was charged to the reactor and degassed at room temperature overnight with nitrogen purge. Then it was stirred and heated to 260 ° C. The EtOH produced is distilled. The reaction was monitored and completed after 5.5 h.

The heat source was removed and the reaction mixture was cooled to 80 ° C and heptane (2,000 L) was charged. The mixture was stirred for 3.0 min. The heptane (6,000 L) was charged to the stirred mixture and stirring was continued overnight. The solids were filtered and washed with heptane (4,000 L) and dried in a vacuum homo at 50 ° C to provide compound 15.

[00118] Example le: ethyl 4-oxo-5- (trif luoromethyl) -lH-guinolin-3-carboxylate (16).

A 5-liter 3-mouth vessel was charged with ethyl 8-chloro-4oxo-5- (trifluoromethyl) -1,4-dihydroquinolin-3-carboxylate 15 (100 g, 0.3 mol), ethanol (1250 mL, 12.5 mL / g) and triethylamine (220 mL, 1.6 mol). The container was then charged with 10% Pd / C g (50% wet) at 5 ° C. The reaction was vigorously stirred under hydrogen atmosphere for 20 hours.⁹C, after which the reaction mixture was concentrated to a volume of approximately 150 mL. The product, ethyl 4-oxo-5 (trifluoromethyl) -lH-quinolin-3-carboxylate 16, in the form

<img file="AR080944A1_D0095.tif" />

of a suspension with Pd / C, it was used directly in the next stage.

[00119] Example ld: 4-oxo-5- (trifluoromethyl) -1,4-dihydroquinolin-3-carboxylic acid (17).

Ethyl 4-OXO-5- (trifluoromethyl) -lH-quinolin-3-carboxylate 16 (58 g, 0.2 mol, crude reaction suspension containing Pd / C) was suspended in NaOH (814 mL of 5 M, 4.1 mol) in a 1 L container with a reflux condenser and heated to 80 ^QC for 18 h, followed by subsequent heating to 100 ^toC for 5 h. The reaction was filtered hot through

Celite to remove Pd / C and Celite was rinsed with NaOH 1

N. The filtrate was acidified to approximately pH 1 to obtain a thick white precipitate. The precipitate was filtered, then rinsed with water and cold acetonitrile. The solid was then dried in vacuo to provide 4-oxo-5 (trifluoromethyl) -1,4-dihydroquinolin-3-carboxylic acid 17 as a white solid (48 g, 92% yield).^:H NMR (400.0

MHz, DMSO-de) δ 15.26 (s, IH), 13.66 (s, IH), 8.98 (s, IH), 8.13 (dd, J = 1, 6, 7.8 Hz , IH), 8,067.99 (m, 2H).

Alternative preparation of intermediate 1: 4-Oxo-5 (trifluoromethyl) -1,4-dihydroquinolin-3-carboxylic acid (17).

cf₃ 0 0

<img file="AR080944A1_D0096.tif" />

i) NaOMe.MeOH,

i) acetic acid

<img file="AR080944A1_D0097.tif" />

OH

N

H

<img file="AR080944A1_D0098.tif" />

<img file="AR080944A1_D0099.tif" />

[00120] Example le: acid 8-chloro-4-oxo-5- (trifluoromethyl)

<img file="AR080944A1_D0100.tif" />

1,4-dihydroquinolin-3-carboxylic (15a)

Ethyl 8-chloro-4-oxo-5 (trifluoromethyl) -1,4-dihydroquinolin-3-carboxylate (15) (1200 g, 3,754 mol) was charged into a reaction vessel followed by the addition of 2-propanol ( l, 200L) and water (7,200 L) and stirred. Sodium hydroxide (600.6 g, 7.508 mol) and water (1,200

L) mixed and allowed to cool to room temperature.

The resulting mixture was charged to the reaction vessel and then heated to 80 ° C and stirred for 3.5 h to generate a homogeneous dark mixture. After one more hour, the acetic acid (9,599 L of 20% w / v, 31.97 mol) was added by means of a drip funnel for 45 min. The reaction mixture was cooled with stirring to 22 ° C at a rate of ° C / h. The resulting solid was filtered and washed with water (3 L) to generate a wet cake (143 6 g). The filtrate was dried in a vacuum homogen with a nitrogen purge on Drierite® to generate 8-chloro-4-oxo-5- (trifluoromethyl) -1,4 dihydroquinolin-3-carboxylic acid in the form of a brown solid (1069

g). Soro doro 4 oxo-5 (trif luoromethyl) -1,4-dihydroquinolin-3-carboxylic acid was purified by suspension in 1.5

L methanol and stirring for 6 h. It was then filtered and dried to obtain 968.8 g of purified 8-chloro-4-oxo-5- (trifluoromethyl) 1,4-dihydroquinolin-3-carboxylic acid.

[00121] Example 1: 4-oxo-5- (trifluoromethyl) -1,4-dihydroquinolin-3-carboxylic acid (17). Compound 15a (18.5 g, 1.00 eq, limited reagent) was charged in

<img file="AR080944A1_D0101.tif" />

a reaction vessel and MeOH (118 mL, 6.4 vol) was added under an inert atmosphere with stirring. Sodium methoxide (3.53 g, 1.00 eq.) Was added portionwise for 10 min to the reactor. The mixture was stirred until all solids were in solution (5-10 minutes). Then palladium on carbon (2.7 g, 0.03 eq) was added to the reaction mixture. Potassium formate (10.78 g, 2 eq.) Dissolved in MeOH (67 mL, 3.6 voi) was added to the reaction mixture for 30 min [alternatively, the potassium formate reagent can be replaced by hydrogen gas). It was then stirred for approximately 4.5 h at room temperature. The reaction was judged complete when the 8-chloro-4-oxo-5- (trifluoromethyl) 1,4-dihydroquinolin-3-carboxylic acid was not more than 1.0% with respect to the 4-oxo-5- (trifluoromethyl acid) ) -1,4-dihydroquinolin3-carboxylic (17). Once the reaction was complete, the mixture was filtered through a plug of Celite (used Celite mass of approximately 2 x mass of 8-chloro-4-oxo-5 (trifluoromethyl) -1,4-dihydroquinolin-3- acid carboxylic charged in the container at the beginning) to remove solids. Cake

Celite was washed with MeOH (37 mL, 2 voi). The filtrate was charged in a clean reaction vessel and stirred. Acetic acid (7.22 mL, 2 eq.) Was continuously charged to the stirred solution for at least 45 minutes and the resulting suspension was stirred for 5-16 h. The solid was filtered and the cake was washed with MeOH (56 mL, 3 voi), dried by suction and then dried in vacuo

<img file="AR080944A1_D0102.tif" />

to give the title compound as an off-white solid.

Intermediary 2: 4- (7-Azabicyclo [2,2,1] heptan-7-yl) -2 (trifluoromethyl) aniline (20).

or₂n

<img file="AR080944A1_D0103.tif" />

cf₃

Γ1

N ^HCI ch₃cn, tea ° C, 16 h

OR₂N

<img file="AR080944A1_D0104.tif" />

cf₃

Pd / C, H₂EtOH, 12 h

H₂N

<img file="AR080944A1_D0105.tif" />

cf₃

7th 19 20

100122]

Example 1 g: 7- [4-Nitro-3- (trifluoromethyl) phenyl] -7azabicyclo [2,2,1] heptane

Method 1 (19) [00123] To a container with 7azabicyclo hydrochloride [2,2,1] heptane 7a (4.6 g, 34.43 mmol, obtained from

Tiger Scientific Inc., 324 Stokes Avenue, Ewing, NJ, 08638

United States, under a nitrogen atmosphere, was added to 4-fluoro-l-nitro-2- (trifluoromethyl) benzene 18 solution (6.0 g, 28.69 mmol) and triethylamine (8.7 g, 12.00 mL, 86.07 mmol) in acetonitrile (50 mL). The reaction vessel was heated at 80 ° C under a nitrogen atmosphere for 16 h. The mixture of

<td>reaction is</td><td>left</td><td>cool</td><td>and</td><td>then I know</td><td>divided</td><td>in</td><td>water and</td>

<td>dichloromethane</td><td>The</td><td colspan="2">organic layer</td><td colspan="2">washed with 1 M</td><td>HCl,</td><td>it dried up</td>

<td>about Na₂SC&gt; 4,</td><td>he</td><td>filtered and</td><td>he</td><td>concentrated</td><td>until</td><td colspan="2">dryness. The</td>

Purification by silica gel chromatography (10% ethyl 0-acetate in hexanes) resulted in 7- [4-nitro-3 (trifluoromethyl) phenyl] -7-azabicyclo [2,2,1] heptane (19) (7.2 g,

88% yield) in the form of a yellow solid. ^TH NMR (400.0

<img file="AR080944A1_D0106.tif" />

MHz, DMSO-dg) δ 8.03 (d, J = 9.1 Hz, 1H), 7.31 (d, J = 2.4 Hz,

<img file="AR080944A1_D0107.tif" />

1H), 7.25 (dd, J = 2.6, 9.1 Hz, 1H), 4.59 (s, 2H), 1.69 - 1.67 (m, 4H), 1.50 (d , J = 7.0 Hz, 4H).

Method 2 [00124] 4-Fluoro-l-nitro-2- (trifluoromethyl) benzene (18) (901 g, 4,309 mol) was introduced into a 30 L jacketed vessel along with Na₂CO₃ (959.1 g, 9.049 mol) and DMSO (5 L, 5.5 vol) under nitrogen atmosphere and stirring. 7azabicyclo hydrochloride [2,2, ljheptane (7a) (633.4 g, 4,740 mol) was then added to the vessel in portions. The temperature rose gradually to 55 ° C. Once the reaction was substantially complete, the mixture was diluted with 10 vol of

EtOAc and washed with water (5.5 vol) three times or until DMSO in the aqueous layer had disappeared (HPLC). The organic layer was concentrated to 4 vol and then the solvent was removed with cyclohexane until all EtOAc was removed and the total volume in the vessel was approximately 4 voi with cyclohexane. The reaction mixture was heated to 60 ° C on a rotary evaporator for 30 min. Then, the solution was cooled to room temperature with stirring or rotation for 3 h.

After the entire solid crystallized, the solution was concentrated to dryness to provide 7- [4-nitro-3- (trifluoromethyl) phenyl] -7-azabicyclo [2,2,1] heptane (19).

Method 3 [00125] 4-Fluoro-l-nitro-2- (trif luoromethyl) benzene (18) was dissolved in 3 voi of DCM. Tetrabutylammonium bromide (0.05 eq)

<img file="AR080944A1_D0108.tif" />

and KOH (50% by weight, 3.6 eq) were added. Then 7-azabicyclo [2,2,1] heptane hydrochloride (7a) was added at 0-5^SC. The reaction was heated to room temperature and monitored with

HPLC Once substantially complete, the layers were separated and the organic layer was washed with 1 M HCl. The layers were separated and the aqueous layer was discarded. The organic layer was washed once with water, once with brine and then distilled. The resulting material was recrystallized from cyclohexane at reflux. The solid was filtered, washed with cyclohexane and dried in a vacuum oven at 45 ° C with a gas purge N₂ to provide 7- [4-nitro-3- (trifluoromethyl) phenyl] -7-azabicyclo [2,2,1] heptane (19).

[00126] Example Ih: 4- (7-Azabicyclo [2,2,1] heptan-7-yl) -2 (trifluoromethyl) aniline (20).

A vessel loaded with 7- [4-nitro-3- (trifluoromethyl) phenyl] -7azabicyclo [2,2,1] heptane 19 (7.07 g, 24.70 mmol) and 10% Pd / C (0.71 g, 6.64 mmol) was evacuated and then flooded with nitrogen.

Ethanol (22 mL) was added and the reaction vessel was equipped with a hydrogen balloon. After stirring vigorously for 12 h, the reaction mixture was purged with nitrogen and the

Pd / C was removed by filtration. The filtrate was concentrated to a dark oil under reduced pressure and the residue was purified by silica gel chromatography (0-15% ethyl acetate in hexanes) to provide 4- (7-azabicyclo [2,2,1] heptan-7-yl) -2 (trifluoromethyl) aniline (20) in the form of a purple solid (5.76 g, 91% yield). 'h NMR (400.0 MHz, DMSO-d₆) ö 6.95 (dd, J

2.3, 8.8 Hz, IH), 6.79 (d, J = 2.6 Hz, IH), 6.72 (d, J = 8.8

Hz, IH), 4.89 s, 2H), 4.09 s, 2H), 1.61

1, 59

<img file="AR080944A1_D0109.tif" />

(d, J = 6.8 Hz, 4H).

Example li: Preparation of sai hydrochloride of 4— (7-azabicyclo [2,2,1] heptan-7-yl) -2- (trifluoromethyl) aniline (20—

HCl).

<img file="AR080944A1_D0110.tif" />

<img file="AR080944A1_D0111.tif" />

Palladium on carbon (150 g, 5% w / w) was charged in a Büchi hydrogenator (20 L capacity) under a nitrogen atmosphere followed by the addition of the 7- [4-nitro-3- sai hydrochloride ( trifluoromethyl) phenyl] -7-azabicyclo [2,2,1] heptane (19) (1500 g) and 2-methyltetrahydrofuran (10.5 L, 7 vol). Hydrogen gas is charged in the closed vessel at a pressure of +0.5 bar above atmospheric pressure. A vacuum was applied for approximately 2 min followed by the introduction of hydrogen gas at a pressure of 0.5 bar. This process was repeated 2 times. Next, hydrogen gas was continuously charged at +0.5 bar above atmospheric pressure. The mixture was stirred and the temperature was maintained between 18 ° C and 23 ° C by cooling the vessel jacket. Once the reaction consumed no more hydrogen and produced no more heat, a vacuum was applied again.

The nitrogen gas was charged into the vessel at 0.5 bar and a vacuum was reapplied followed by a second load of 0.5 bar of nitrogen gas. Once the reaction is substantially complete, the

<img file="AR080944A1_D0112.tif" />

Reaction mixture was transferred to a receiving vessel under nitrogen atmosphere by means of a filter funnel using a Celite filter. The Celite filter cake was washed with 2 methyltetrahydrofuran (3 L, 2 voi). The washings and the filtrate were loaded in a container equipped with stirring, temperature control and a nitrogen atmosphere. 4M HCl was added in

1,4-dioxane (1 voi) continuously for 1 h in a vessel at 20 ° C. The mixture was stirred for a further 10 h (or filtered and overnight), washed with 2 methyltetrahydrofuran (2 vol) and dried to generate 1519 g of (trifluoromethyl) aniline hydrochloride

4- (7-azabicyclo [2,2,1] heptan-7-yl) -2 [20-HCl) in the form of a white crystalline solid.

Example lj: Preparation of compound 1.

<img file="AR080944A1_D0113.tif" />

20 1

100127] To a solution of 4-oxo-5- (trifluoromethyl) -1Hquinolin-3-carboxylic acid 17 (9.1 g, 35.39 mmol) and 4- (7azabicyclo [2,2,1] heptan-7- il) -2- (trifluoromethyl) aniline 20 (9.2 g, 35.74 mmol) in 2-methyltetrahydrofuran (91.00 mL) cyclic propylphosphonic acid anhydride (T3P, 50% solution in ethyl acetate, 52 was added) , 68 mL, 88.48 mmol) and pyridine (5.6 g, 5.73 mL, 70.78 mmol) at room temperature. The vessel of

<img file="AR080944A1_D0114.tif" />

The reaction was heated at 65 ° C for 10 h under a nitrogen atmosphere. After cooling to room temperature, the reaction was diluted with ethyl acetate and neutralized with saturated Na solution.₂CO₃ (50 mL). The layers were separated and the aqueous layer was extracted twice more with ethyl acetate. The combined organic layers were washed with water, dried over

Na₂SC&gt; 4, filtered and concentrated on a roasted solid. The crude solid product was suspended in ethyl acetate / diethyl ether (2: 1), collected by vacuum filtration and washed

<td colspan="2">twice more with acetate</td><td>from</td><td>ethyl / ether</td><td>diethyl</td><td>(2: 1) for</td>

<td>provide the</td><td>product</td><td>in</td><td>form of</td><td>a dust</td><td>crystalline</td>

<td>darò yellow.</td><td>Dust</td><td>he</td><td>dissolved</td><td>in acetate</td><td>of ethyl</td>

<td>hot and it</td><td>absorbed</td><td>in</td><td>Celite</td><td colspan="2">Purification by</td>

<td>chromatography on</td><td>gel of</td><td colspan="2">silica (0-50%</td><td>acetate</td><td>ethyl in</td>

<td>dichloromethane)</td><td>gave N- (</td><td> :4-(7</td><td colspan="3">-azabicyclo [2,2,1] heptan-7-yl) -2-</td>

(trifluoromethyl) phenyl) -4-oxo-5- (trifluoromethyl) -1,4dihydroquinolin-3-carboxamide in the form of a crystalline solid

<td>bianco</td><td> (13</td><td>, 5 g,</td><td> 76%</td><td>from</td><td colspan="2">performance)</td><td colspan="2">. LC / MS m / z 496.0 [M + H] *,</td>

<td>weather</td><td>from</td><td colspan="2">retention</td><td> 1,48</td><td>min</td><td>(rp-c₁₈,</td><td>10-99% CH₃CN / 0.05%</td><td>TFA</td>

<td>during</td><td> 3</td><td>min)</td><td>³Η</td><td>NMR</td><td> (400,0</td><td>MHz,</td><td>DMSO-d₆) δ 13.08 (s,</td><td>1 HOUR) ,</td>

<td> 12,16 (</td><td>s,</td><td>IH),</td><td> 8, 88</td><td>(s,</td><td>IH),</td><td> 8,04 (</td><td>dd, J = 2.1, 7.4 Hz,</td><td>1 HOUR) ,</td>

<td> 7,95 -</td><td colspan="2">7.88 (m,</td><td>3H),</td><td colspan="2">7.22 (dd,</td><td> 2,5,</td><td>8.9 Hz, IH), 7.16 (d,</td><td>J =</td>

<td>2.5 Hz,</td><td>IH</td><td colspan="2">), 4.33 (s,</td><td>, 2H)</td><td> , 1,67</td><td>(d, J</td><td>= 6.9 Hz, 4H), 1.44 (d,</td><td>J =</td>

<td>6.9 Hz,</td><td>4h;</td><td> 1 .</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> [00128]</td><td colspan="2">Synthesis</td><td>from</td><td colspan="3">hydrochloride</td><td colspan="2">7-azabicyclo [2,2, ljheptane</td>

(7a).

<img file="AR080944A1_D0115.tif" />

starting material: trans-4-aminoecyclohexanol

TFA

TFA

-►

Aqueous NaOH, then HCI (gas)

-► • HCI 2

OM 3 c

7a [00129] Example 1k: Preparation butoxycarbonylamino) cyclohexane1 of trans-4- (ter (A).

Method 1

[00130] Sodium carbonate (920.2 g, 8.682 mol, 2 eq) was added to a reaction vessel followed by the addition of water (3,000

L, 6 vol) and stirring. Dichloromethane (DCM, 4,000 L, 4 vol) was added followed by trans-4-aminocyclohexanol (500.0 g, 4,341 mol) to generate a biphasic reaction mixture that was vigorously stirred at room temperature. A solution of Boc₂0 (947.4 g, 997.3 mL, 4,341 mol, 1 eq) in DCM (2 voi) was then quickly added dropwise to the vessel and the resulting reaction mixture was stirred at room temperature overnight. The reaction mixture was then filtered and the filter cake washed with water (2x8 voi). The product was dried by suction until it was a compact cake. The cake was then dried in a vacuum oven at 35 ° C for 2 4 h giving 83 0 g of (A) as a

<img file="AR080944A1_D0116.tif" />

trans-4- (tert-butoxycarbonylamino) cyclohexanol solid crystalline.

Method 2

<img file="AR080944A1_D0117.tif" />

[00131] Two containers with a round base of 50 L of three mouths were each equipped with a mechanical stirrer and thermocouple. The containers were placed in a cooling tube and then each container was charged with water (8.87 L) and trans-4-aminocyclohexanol (1479 g). After about 10 to 30 minutes, the trans-4-aminocyclohexanol had dissolved and potassium carbonate (1774.6 g) was added to each vessel.

After about 10 to 20 minutes, the potassium carbonate had dissolved and DCM (2.96 L) was charged to each vessel. Boc anhydride (3082.6 g) in DCM (1479 mL) was then added to each vessel at such speed as to maintain the temperature at 20-30 ° C. An ice / water bath was used to control the exotherm and to accelerate the addition that took approximately 1 to 2 hours. A suspension was formed during the addition and the reaction mixtures were allowed to warm to room temperature and stirred overnight, until the reaction was completed based on the disappearance of Boc anhydride. Heptane (6 L) was then charged to each vessel and the mixtures were cooled to about 0-5 ° C. The solids were collected from each vessel by filtration using the same filter. The combined solids were washed with heptane (6

L) followed by water (8 L). The solids were loaded into an appropriate size container equipped with a mechanical stirrer. Water (12 L)

<img file="AR080944A1_D0118.tif" />

and heptane (6 L) were added and the resulting suspension was added mechanically for 30 to 60 minutes. The solids were collected by filtration and then washed on a filter with water (8 L) and heptane (8 L), air dried on a filter for three days and then dried under vacuum at 30 to 35 ° C to weight. constant to provide the product in the form of a solid bianco.

[00132] Example 11: Preparation of trans-4- (terbutoxycarbonylamino) cyclohexylmethanesulfonate (B).

Method 1

[00133] A 12 L vessel was equipped with a nitrogen flow and a mechanical stirrer. Trans-4- (terbutoxycarbonylamino) cyclohexanol (750 g, 3,484 mol) was introduced, followed by tetrahydrofuran (THF, 6,000 L, 8 vol) and the mixture was stirred. Triethylamine (370.2 g, 509.9 mL, 3.658 mol,

1.05 eq) was added and the mixture was cooled to 0 ° C. Methanesulfonyl chloride (419.0 g, 283.1 mL, 3.688 mol, 1.05 eq) were carefully added dropwise, keeping the temperature of the mixture at less than 5 ° C. After the addition, the mixture was stirred at 0 ° C for 3 h and then gradually heated to room temperature (17 ° C) and stirred overnight (approximately 15 h). The mixture was neutralized with water (6 vol) and stirred for 15 min. Ethyl acetate (EtOAc, 9,000 L, 12 vol) was added and stirring was continued for 15 min. Stirring was stopped and the mixture was allowed to stand for 10 min and the aqueous phase was removed. 1 N HCl (6 vol, 4.5 L) was added and stirring was continued for 15 min. The agitation stopped and

<img file="AR080944A1_D0119.tif" />

The aqueous phase was removed. 10% w / v NaHCO₃ (4.5 L, voi)

<td>added</td><td>and</td><td>the</td><td>mixture</td><td>he</td><td>agitò</td><td>for 10 min</td><td>Agitation</td><td>he</td>

<td>stopped</td><td>and</td><td>the</td><td colspan="3">aqueous phase is</td><td>eliminated. Water (</td><td>6 voi, 4,5 L)</td><td>he</td>

<td>added</td><td>and</td><td>the</td><td>mixture</td><td>he</td><td>agitò</td><td>for 10 min.</td><td>The aqueous layer</td><td>he</td>

removed and the organic layer was filtered and concentrated to 4 voi. Heptane (5.5 voi, 4 L) was added and the mixture was concentrated again to dryness resulting in 988 g of trans-4- (terbutoxycarbonylamino) cyclohexylmethanesulfonate.

Method 2

100134] A three-round round base vessel equipped with a mechanical stirrer, addition funnel, nitrogen inlet, thermocouple and drying tube was placed in a cooling tube. Trans-4- (tert-butoxycarbonylamino) cyclohexanol (2599 g, 12.07 moi, 1.0 eq), tetrahydrofuran (THF) (20.8 L) and triethylamine (1466 g, 14.49 moi, 1.2 eq ) were added to the container. The mixture was cooled with a bath of ice water and stirred. Methanesulfonyl chloride (1466 g, 12.80 moi, 1.06 eq) was added dropwise by means of the addition funnel for 1 hour. Once the addition was complete, the cooling barium was removed and the reaction mixture was stirred until TLC indicated that the starting material had been consumed (approximately minutes). The reaction mixture was then neutralized with an aqueous solution of hydrochloric acid (223 mL of HCl in 6.7 L of water) and EtOAc (10.4 L). The mixture was stirred for approximately 10 to 20 minutes at room temperature and then transferred to a separatory funnel. The layers separated

<img file="AR080944A1_D0120.tif" />

and the aqueous layer was discarded. The organic layer was washed with water (2 x 4.5 L), saturated aqueous sodium bicarbonate solution (1 x 4.5 L) and dried over anhydrous magnesium sulfate with stirring for 5 to 10 minutes. The mixture was filtered and the filter cake was washed with EtOAc (2 x 600 mL). The combined washes and the filtrate were concentrated under reduced pressure at 40 ° C, giving a white solid. The solid was extracted into heptane (3 L) and cooled in an ice / methanol cooling tube. More heptane (5 L) was added and the mixture was stirred at 0 to 5 ° C for not less than 1 hour. The solids were then collected by filtration, washed with cold heptane (0 to 5 ° C, 2 x 1.3 L) and dried in vacuo at 40 ° C to a constant weight to provide the product.

[00135] Note: A jacketed reactor can be used instead of a round base vessel with a cooling tube and ice bath.

[00136] Example lm: Preparation of trans-4-aminocyclohexylmethanesulfonate (C).

Method 1

[00137] Trans-4- (tert-butoxycarbonylamino) cyclohexymethanesulfonate (985 g, 3,357 moi) was introduced into a 12 L vessel of three mouths equipped with an agitator under an atmosphere of nitrogen and ventilation. DCM (1,970 L, 2 voi) was added at room temperature and stirring began. Trifluoroacetic acid (TFA) (2,844 kg, 1,922 L, 24.94 moi, 2 voi) was added slowly to the mixture in two batches of 1 L each. After the first

<img file="AR080944A1_D0121.tif" />

Second addition The mixture was stirred overnight (15 h) at room temperature resulting in a clear solution. 2 Methyltetrahydrofuran (4 voi) was then added to the reaction mixture, which was stirred for 1 h. The mixture was then carefully filtered in a fume hood and dried by suction to generate 1100 g of trans-4-aminocyclohexylmethanesulfonate TFA sai with excess TFA.

Method 2

[00138] A 50 L round base vessel of three mouths was equipped with a mechanical stirrer, addition funnel and thermocouple and placed in a cooling tube. To the vessel trans-4- (tert-butoxycarbonylamino) cyclohexymethanesulfonate (3474 g, 1.0 eq) and DCM (5.9 L) were added to the vessel. The resulting suspension was stirred for 5 to 10 minutes at room temperature and then trifluoroacetic acid (TFA, 5.9 L) was added by adding funnel slowly for 2.5 hours to control the resulting exotherm and the rate of evolution. of gas The reaction mixture was stirred at room temperature overnight and then cooled to 15 ° C to 20 ° C using a barium of ice water. 2-Methyl tetrahydrofuran (2-MeTHF, 11.8 L) was then added by means of an addition funnel at such rate to maintain the internal temperature at less than 25 ° C (approximately 1.5 hours). The addition of the first 4-5 L of

2-MeTHF was exothermic. The resulting suspension was stirred for 1 hour. The solids were collected by filtration and then

<img file="AR080944A1_D0122.tif" />

The product with a white solid was washed with 2-MeTHF (2 x 2.2 L) and room temperature to a weight.

[00139] Example In: Preparation azabicyclo [2,2,1] heptane (7a).

Method 1

Όι

C ·; ' &gt;

ω then they dried up

<img file="AR080944A1_D0123.tif" />

constant to provide 7 [00140] hydrochloride The TFA sai of trans-4-aminocyclohexylmethanesulfonate (200 g, 650.9 mmol) was introduced into a 3-liter vessel followed by the addition of water (2,200 L, 11 voi). NaOH (78.11 g, 1,953 moi, 3 eq) was added slowly, keeping the temperature of the reaction mixture at less than 25 ° C and the mixture was stirred overnight. DCM (1.4 L, 7 voi) was then added and the mixture was stirred and the organic layer separated. The aqueous layer was then extracted a second time with DCM (1.4 L, 7 vol) and the DCM layers were combined. HCI (108.5 mL, 12M,

1.3020 moi, 2 eq) was then added, the mixture was stirred for 30 min and then concentrated on a rotary evaporator to dryness. Acetonitrile (10 voi) was added and the mixture was concentrated. This was repeated three times until all traces of water were removed azeotropically, to provide 7-azabicyclo hydrochloride [2,2,1] heptane (7a). The crude product was recrystallized from acetonitrile (10 voi) to provide 7-azabicyclo [2,2,1] heptane hydrochloride (7a) as a colorless crystalline solid. ¹HRMN (DMSO-d⁶) ppm 8.02-8.04 (d);

7.23-7.31 (m); 4.59 (s); 3.31 (s); 2.51-3.3 (m); 1.63-1.75 (m);

1.45-1.62 (m).

<img file="AR080944A1_D0124.tif" />

[00141] As the product ° C at 97 ° C and observation, instead of adding crude it can also be distilled and then recrystallized.

DCM for extraction at approximately

Method 2

[00142] A round base vessel with three 50 L mouths equipped with a mechanical stirrer, addition funnel and thermocouple and placed in a heat blanket. Trans-4-aminocyclohexylmethanesulfonate trifluoroacetate (3,000 g, 1 eq) and water (30 L) were added to the vessel. The mixture was stirred, as 50% NaOH (2343 g, 29.29 moi, 3 eq) was added with an addition funnel at such a rate to maintain the temperature at less than 25 ° C because the addition was slightly exothermic. After completing the addition of NaOH, the reaction mixture was stirred overnight at room temperature. The product was recovered by distillation by fractionation at reflux temperature (approximately 100 ° C) with a temperature greater than 95 to 98 ° C. The pH of each fraction was adjusted to 2 by the addition of HCl and concentrated under reduced pressure at 55 ° C to leave a thick paste. Acetonitrile (1.5 L ACN) was added and the resulting suspension was stirred for 30 minutes and then cooled to 0 to 5 ° C for 1 hour. The solids were collected by filtration, washed with ice cold ACN (2 x 600 mL) (0 to 5 ° C) and dried in vacuo at 50 ° C to a constant weight.

[00143] A 22 L round base vessel of three mouths was equipped with a mechanical stirrer, thermocouple and condenser and placed in a heat blanket. The solids collected (2382 g),

<img file="AR080944A1_D0125.tif" />

Methanol (4.7 L) and 2-MeTHF (4.7 L) were added to the vessel.

The resulting suspension was stirred and heated to reflux (approximately 65 ° C). The reaction vessel was transferred to a cooling tube and the mixture was stirred. 2-MeTHF (4.7 L) was then added by means of addition funnel for 30 minutes. The resulting suspension was cooled to 0 to 5 ° C and stirred at this temperature for 30 minutes. The solids were collected by filtration, washed with 2-MeTHF (2 x 600 mL) ice cream (0 to 5 ° C) and then dried in vacuo at 55 ° C to a constant weight.

[00144] A 12 L round base vessel of three round mouths equipped with a mechanical stirrer, thermocouple, nitrogen inlet and condenser was placed in a heat blanket. The crude product (2079 g) and ACN (6.2 L) were added to the vessel. The resulting suspension was stirred and heated to reflux (approximately 82 ° C) for 30 minutes. The vessel was transferred to a cooling tube and the suspension was slowly cooled to 0 to 5 ° C and maintained at this temperature for 1 hour. The solids were collected by filtration, washed with ice cold ACN (3 χ 600 mL) (0 to 5 ° C) and dried in vacuo at 55 ° C to a constant weight to achieve the product.

<img file="AR080944A1_D0126.tif" />

Formula II

II.B.1. Embodiments of the compounds of the formula il [00145] In one embodiment, in the compound of the formula II of the composition,

T is -CH₂-, -CH₂CH₂-, -CF₂-, -C (CH₃)₂- O -C (O) -;

R¹ 'is H, aliphatic halo, CF₃, CHF₂, O (aliphatic C₃-₆) ;

and

R^m or R^:32 it's z^dr₉ where :

Z^D It's a link, CONH, SO₂NH, SO₂N (Ci-6 alkyl),

CH₂NHSO₂, CH₂N (CH₃) SO₂, CH₂NHCO, COO, SO₂ or CO; and

Rg is H, aliphatic Ci_₆ or aryl.

II.B.2. Compound 2 [00146] In another embodiment, the compound of formula II is compound 2, shown below, which is also known by its acidic chemical name 3- (6- (1- (2,2di fluorobenzo [d ] [1,3] dioxol-5-yl) cyclopropancarboxamido) -3-methylpyridin-2-yl) benzoic.

<img file="AR080944A1_D0127.tif" />

compound 2

II.B.3. Summary of the syntheses of compound 2 [00147] Compounds of the formula II, such as exemplified by means of compound 2, can be prepared by coupling an acid chloride residue with an amine residue according to the following schemes 2-la at 2-3.

Scheme 2-la: Synthesis of the rest of acid chloride.

<img file="AR080944A1_D0128.tif" />

one. reduction

two. XaOH

<img file="AR080944A1_D0129.tif" />

<img file="AR080944A1_D0130.tif" />

one. NaCN

two. H₂OR

X⁰'

F 0 '

<img file="AR080944A1_D0131.tif" />

NaOH

-atOH

<img file="AR080944A1_D0132.tif" />

<img file="AR080944A1_D0133.tif" />

Koh

<img file="AR080944A1_D0134.tif" />

<img file="AR080944A1_D0135.tif" />

[00148] Scheme 2-la represents the preparation of 1- (2,2-difluorobenzo [d] [1,3] dioxol-5yl) cyclopropanecarbonyl chloride, which is used in Scheme 3 to prepare the amide ligation of the compound 2.

ω [00149] Acid starting material

2,2-

<img file="AR080944A1_D0136.tif" />

difluorobenzo [d] [1,3] dioxol-5-carboxylic, is available at stores in Saltigo (an affiliate of Lanxess Corporation). Reduction of the remaining carboxylic acid in 2,2difluorobenzo [d] [1,3] dioxol-5-carboxylic acid in the primary alcohol, followed by conversion into the corresponding chloride using thionyl chloride (SOCI2), provides 5- ( chloromethyl) 2,2-difluorobenzo [d] [1,3] dioxol, which is then converted to 2 (2,2-difluorobenzo [d] [1,3] dioxol-5-yl) acetonitrile using sodium cyanide. Treatment of 2- (2,2difluorobenzo [d] [1,3] dioxol-5-yl) acetonitrile with base and 1bromo-2-chloroethane provides 1- (2,2difluorobenzo [d] [1,3] dioxol-5 -il) cyclopropanecarbonitrile. The nitrile moiety in 1- (2,2-difluorobenzo [d] [1,3] dioxol-5yl) cyclopropanecarbonitrile is converted to a carboxylic acid using base to give 1- (2,2-difluorobenzo [d] [1 , 3] dioxol-5yl) cyclopropancarboxylic acid, which is converted to the desired acid chloride using thionyl chloride.

2-lb scheme. Alternative synthesis of the rest of acid chloride

<img file="AR080944A1_D0137.tif" />

N HCl DMSO, 'f -5^KC

<img file="AR080944A1_D0138.tif" />

NaOH

BwNBr

SOCI,

-&gt;

<img file="AR080944A1_D0139.tif" />

[00150] Scheme 2-lb provides an alternative synthesis of the required acid chloride. The 5-bromomethyl-2,2-difluoro-1,3-benzodioxole compound is coupled with ethyl cyanoacetate in the presence of a palladium catalyst to form the corresponding cyanoethyl alpha ester. Saponification of the ester moiety in the carboxylic acid gives the cyanoethyl compound. Alkylation of the cyanoethyl compound with 1-bromo-2-chloroethane in the presence of a base gives the cyanocyclopropyl compound. The treatment of the cyanocyclopropyl compound with a base gives the carboxylate sai, which is converted into the carboxylic acid by acid treatment. The conversion of the carboxylic acid into the acid chloride is carried

Cu ;,

<img file="AR080944A1_D0140.tif" />

then carried out using a taies corno chlorination agent dionionyl chloride or the like.

Scheme 2-2: Synthesis of the rest of the amine.

<img file="AR080944A1_D0141.tif" />

CO₂tßu

<img file="AR080944A1_D0142.tif" />

one. K_?CO₅. Pd (dppf) Cl₂

<img file="AR080944A1_D0143.tif" />

urea-hydrogen peroxide phthalic anhydride EtOAc, water

<img file="AR080944A1_D0144.tif" />

CO₂tBu [00151] Scheme 2-2 represents the preparation of the required tert-butyl 3- (6-amino-3-methylpyridin-2-yl) benzoate, which is coupled with 1- (2,2-difluorobenzo [d] chloride) [1,3] dioxol-5il) cyclopropanecarbonyl in Scheme 3 to give the compound

two. Coupling of 2-bromo-3-methylpyridine with palladium catalyzed 3- (terbutoxycarbonyl) phenylboronic acid gives tert-butyl 3- (3-methylpyridin-2-yl) benzoate, which then becomes the desired compound.

<img file="AR080944A1_D0145.tif" />

Scheme 2-3: Formation of a 3- (6- (1- (2,2-difluorobenzo [d] £ 1,3] dioxol-5-yl) cyclopropancarboxamido) -3-methylpyridin-2-yl) benzoic acid sai acid.

<img file="AR080944A1_D0146.tif" />

<img file="AR080944A1_D0147.tif" />

• acid

TO

CO-tBu [00152] Scheme 2-3 represents the coupling of 1- (2,2-difluorobenzo [d] [1,3] dioxol-5-yl) cyclopropanecarbonyl chloride with 3- (6-amino-3- tert-butyl methylpyridin-2-yl) benzoate using triethylamine and 4-dimethylaminopyridine to initially provide the tert-butyl ester of compound 2. Treatment of the tert-butyl ester with an acid taies as HC1 gives the HCl sai of compound 2, which is typically a crystalline solid.

II.B.4. Examples: Synthesis of compound 2 [00153] Vitride® (bis (2-methoxyethoxy) aluminum and sodium hydride [or NaAlH₂ (OCH2CH2OCH3) 2], 65% solution by weight in toluene) was purchased from Aldrich Chemicals. 2,2Difluoro-1,3-benzodioxol-5-carboxylic acid was purchased from Saltigo (an affiliate of Lanxess Corporation).

[00154] Example 2a: (2,2-Difluoro-1,3-benzodioxol-5-yl) methanol.

<img file="AR080944A1_D0148.tif" />

<img file="AR080944A1_D0149.tif" />

-OH

<img file="AR080944A1_D0150.tif" />

CO-H

Vitride (2 equiv)

PhCH; (10 vol)

10% aq (ww) NaOH (4 equiv Ì

F '

86-92% 9e ænd [00155] Commercially available 2,2-dif luoro-1,3-benzodioxol-5-carboxylic acid (1.0 eq) was suspended in toluene (10 vol). Vitride® (2 eq) was added by means of addition funnel at such a speed as to maintain the temperature at 15-25 ° C.

At the end of the addition, the temperature was raised to 40 ° C for hours (h), then 10% aqueous NaOH (w / w) (4.0 eq) was carefully added by means of addition funnel, maintaining the temperature at 40-50 ° C. After stirring for 30 minutes (min) more, the layers were allowed to separate at 40 ° C. The organic phase was cooled to 20 ° C, then washed with water (2 x 1.5 voi), dried (Na₂SW₄), was filtered and concentrated to obtain crude (2,2-difluoro-1,3-benzodioxol-5-yl) -methanol which was used directly in the next step.

[00156] Example 2b: 5-Chloromethyl-2,2-dif luoro-1,3-benzodioxol.

one. SOCI-. (1.5 equiv)

DMAP (0.01 equiv)

MTBE (5 voi)

two. water (4 voi)

<img file="AR080944A1_D0151.tif" />

OH

82-100 ° or rend.

100157] (2,2-Difluoro-1,3-benzodioxol-5-yl) -methanol (1.0 eq) was dissolved in MTBE (5 voi). A catalytic amount of 4- (N, Ndimethyl) aminopyridine (DMAP) (1 moi%) was added and SOC1₂ (1.2 eq)

<img file="AR080944A1_D0152.tif" />

was added by means of addition funnel. The SOC1₂ was added to^v such a speed as to maintain the temperature in the reactor at 15-25 ° C. The temperature rose to 30 ° C for h and then cooled to 20 ° C. Water (4 voi) was added by means of addition funnel while maintaining the temperature at less than 3 ° C. After stirring for 3.0 min more, the layers were separated. The organic layer was stirred and 10% aqueous NaOH (w / v) (4.4 voi) was added. After stirring for 15 to 20 min, the layers could be separated. The organic phase then dried up (Na2SO₄), was filtered and concentrated to obtain crude 5-chloromethyl-2,2-difluoro-1,3-benzodioxol which was used directly in the next step.

[00158] Acetonitrile example.

2 C :

(2,2-Difluoro-1,3-benzodioxol-5-yl) -

<img file="AR080944A1_D0153.tif" />

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

two. water (6 voi)

Μ 1BE (4 voi)

95-100 ° or rend.

χΧχ η - .. ί '

F. Ο

F Ο

-C Ν [00159] A solution of 5-chloromethyl-2,2-difluoro-1,3benzodioxol (1 eq) in DMSO (1.25 voi) was added to a suspension of NaCN (1.4 eq) in DMSO ( 3 voi), while maintaining the temperature between 30-40 ° C. The mixture was stirred for 1 h and then the water (6 voi) was added, followed by methyl ether butyl ether (MTBE) (4 voi). After stirring for 30 min, the layers were separated. The aqueous layer was extracted with MTBE (1.8 voi). The combined organic layers were washed with water (1.8 vol), dried (Na₂SW₄), filtered and concentrated to obtain crude (2,2-difluoro-1,3-benzodioxol-5-yl) -acetonitrile (95%) which was used directly in the next step. ^]H NMR (500 MHz, DMSO) δ 7.44 (br s, 1H), 7.43 (d, J = 8.4 Hz, 1H), 7.22 (dd, J = 8.2, 1.8 Hz, 1H), 4.07 (s, 2H).

[00160] Example 2d: Alternative synthesis of (2,2-dif luoro-1,3benzodioxol-5-yl) -1-ethyl acetate-acetonitrile

<img file="AR080944A1_D0154.tif" />

you

EtO

Pd (dba) i. aBu; P -—Na_sPO ·, toluene_: H 0 ^:C

F-.

xj: ia

F 0 '-u- γ _0Et

CN [00161] A reactor was purged with nitroqen and charged with toluene (900 mL). The solvent was degassed by means of nitroqenne purqa for not less than 16 hours. Lueqo Na was loaded into the reactor₃PO₄ (155.7 q, 949.5 mmol), bis (dibenzylidenacetone) palladium (0) dry (7.28 g, 12.66 mmol). A 10% w / w solution of tert-butylphosphine in hexanes (51.23 q,

25.32 mmol) was charged for 10 minutes at 23 ^?Addition funnel C purged with nitroqen. The mixture was left under stirring for 50 minutes, at which time 5-bromo-2,2-difluoro-1,3-benzodioxol (75 g, 316.5 mmol) was added for 1 minute.

After stirring for 50 minutes more, the mixture was charged with ethyl cyanoacetate (71.6 g, 633.0 mmol) for 5 minutes, followed by water (4.5 mL) in one portion. The mixture was heated to 70^PC for 40 minutes and analyzed by HPLC every 1 to 2 hours for the percentage of conversion of the reagent into the product. After observing the complete conversion

<img file="AR080944A1_D0155.tif" />

(typically 100% conversion after 5 to 8 hours), the mixture was cooled to 20 to 25 ²C and filtered through a celite cue. The celite taco was rinsed with toluene (2 X

450 mL) and the combined organic layers were concentrated in 300 mL in vacuo at 60 to 65 ^toC. The concentrate was charged with DMSO (225

<td>mL) and concentrated on</td><td>empty of</td><td> 70</td><td>to 80 ²C</td><td>see you later</td><td>cesò</td><td>the</td>

<td>active distillation of</td><td>solvent.</td><td>The</td><td>solution</td><td>He cooled</td><td>until</td><td> 20</td>

<td>to 25 ^SC and diluted in</td><td>900 mL</td><td>with</td><td>DMSO in</td><td>preparation</td><td>for</td><td>the</td>

stage 2. ^!H NMR (500 MHz, CDC1₃) ò 7.16 - 7.10 (m, 2H), 7.03 (d,

J = 8.2 Hz, IH), 4.63 (s, IH), 4.19 (m, 2H), 1.23 (t, J - 7.1

Hz, 3H).

[00162] Example 2e: Alternative synthesis of (2,2-difluoro-1,3-benzodioxol-5-yl) -acetonitrile.

^Fx ° ïi S ^3Nhcl

F DMSO, 75 ° CF

CN [00163] The DMSO solution of (2,2-difluoro-1,3-benzodioxol5-yl) -1-ethyl acetate-acetonitrile above was charged with HCI

N (617.3 mL, 1.85 moi) for 20 minutes while maintaining an internal temperature below 40 ^andC. The mixture was then heated to 75 ²C for 1 hour and analyzed by HPLC every 1 hours for the conversion rate. When a conversion greater than 99% was observed (typically after 5 to 6 hours), the reaction was cooled to 20 to 25 C and extracted with

MTBE (2 X 525 mL), with enough time to allow full phase separation during extractions. The combined organic extracts were washed with 5% NaCl (2 X 375

<img file="AR080944A1_D0156.tif" />

mL) The solution was then transferred to an appropriate equipment for a distillation at 1.5-2.5 of vacuum which was equipped with a cooled receiver vessel. The solution was concentrated in vacuo to less than 60^SC to remove solvents. (2,2-Difluoro-1,3benzodioxol-5-yl) -acetonitrile is distilled after the resulting oil of 125 to 130²C (homo temperature) and 1.5 to 2.0

Torr. (2,2-Dìfluoro-1,3-benzodioxol-5-yl) -acetonitrile was isolated as a darò oil with a 66% yield of 5-bromo-2,2difluoro-1,3-benzodioxol (2 steps) and with an HPLC purity of

<td>from AUC</td><td>(corresponds</td><td>yet</td><td>test</td><td>p / p</td><td>of 95%). ³Η NMR</td><td> (500</td>

<td>DMSO) δ</td><td>7.44 (br s,</td><td>IH),</td><td> 7,43 (</td><td>d, J</td><td>= 8.4 Hz, IH),</td><td> 7,22</td>

<td>J = 8.2,</td><td>1.8 Hz, IH),</td><td> 4,07</td><td>(s, 2H)</td><td></td><td></td><td></td>

(2,2-Difluoro-1,3-benzodioxol-5-yl) [00164] Example 2f:

cyclopropanecarbonitrile.

3-Bromo-2 - Chloroethane (1.5 equiv) 50% KOH (5.0 equiv)

Oct.NBr (0.02 equiv)

<td>xxx, „</td><td> “0</td><td>degrees C H.H</td><td> 5-100%</td><td>of rend.</td><td>χχ&gt;_Δ.,,</td>

<td>[00165] One</td><td colspan="2">mixture</td><td>from</td><td>(2,2-Difluoro-</td><td>• 1,3-benzodioxole-5-yl) -</td>

<td>acetonitrile '</td><td> (1,0</td><td>eq),</td><td>Koh</td><td>50% aqueous</td><td>by weight (5.0 eq), 1-</td>

<td colspan="2">bromine-2-chloroethane</td><td> (1,5</td><td>eq)</td><td colspan="2">and Oct₄NBr (0.02 eq) was heated to 70</td>

<td>° C for 1</td><td>h.</td><td colspan="2">Mix</td><td>of reaction</td><td>it cooled down then</td>

elaborated with MTBE and water. The organic phase was washed with water and brine. The solvent was removed to obtain (2,2-difluoro-1,3-benzodioxole-5-yl) -cyclopropanecarbonitrile. H NMR (500 MHz,

<img file="AR080944A1_D0157.tif" />

DMSO) δ 7.43 (d, J = 8.4 Hz, 1H), 7.40 (d, J = 1.9 Hz, 1H), 7.

<img file="AR080944A1_D0158.tif" />

(dd, J = 8.4, 1.9 Hz, 1H), 1.75 (m, 2H), 1.53 (m, 2H).

[00166] Example 2 g: 1- (2,2-Difluoro-1,3-benzodioxol-5-yl) -cyclopropancarboxylic acid.

M NaOH (8 equiv) EtOH (5 vol). 80 degrees C MTBE (10 vol! Dicyclohexylamine (1 equiv)

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

ΌΗ

69% yield [00167] (2,2-Difluoro-1,3-benzodioxol-5-yl) cyclopropanecarbonitrile was hydrolyzed using 6 M NaOH (8 equiv) in ethanol (5 vol) at 80 ° C overnight. The mixture was cooled to room temperature and the ethanol was evaporated in vacuo. The residue was extracted in water and MTBE, 1 M HCl was added and the layers were separated. The MTBE layer was then treated with dicyclohexylamine (DCHA) (0.97 equiv). The suspension was cooled to 0 ° C, filtered and washed with heptane to give the corresponding sai of DCHA. The sai was extracted in MTBE and 10% citric acid and stirred until all solids had dissolved. The layers were separated and the MTBE layer was washed with water and brine. An imbibition of solvent in heptane followed by filtration gave 1- (2,2-difluoro-1,3-benzodioxol5-yl) -cyclopropancarboxylic acid after drying in a vacuum homo at 50 ° C overnight. ESI-MS m / z cale. 242.04, found

241.58 (M + l)⁴; ^:H NMR (500 MHz, DMSO) δ 12.40 (s, IH), 7.40 (d, J

<img file="AR080944A1_D0159.tif" />

= 1.6 Hz, IH), 7.30 (d, J = 8.3 Hz, 1H), 7.17 (dd, J = 8.3, 1.7

Hz, 1H), 1.46 (m, 2H), 1.17 (m, 2H).

[00168] Example 2h: 1- (2,2-Difluoro-1,3benzodioxol-5-yl) -cyclopropancarbonyl chloride.

F ⁰

X

FO

<img file="AR080944A1_D0160.tif" />

SOCI-.,

PhCHn degrees C

F·,.

XI [00169] 1- (2,2-Difluoro-1,3-benzodioxol-5-yl) cyclopropancarboxylic acid (1.2 eq) was suspended in toluene (2.5 vol) and the mixture was heated to 60 ° C . SOCI ?. (1.4 eq) was added by means of addition funnel. Toluene and SOC1₂ they were distilled from the reaction mixture after 30 minutes. Additional toluene (2.5 vol) was added and the resulting mixture was distilled again, giving the product of acid chloride as an oil, which was used without further purification.

[00170] Example 2i: terbutyl 3- (3-methylpyridin-2-yl) benzoate.

Il I 'N Br

CO-tBu

one. toluene, 2 \ 1 KiCO?

Pdf dppf iCh. S0 degrees C

MsOH aquoio

Aqueous XaOH

CT: odBu [00171] 2-Bromo-3-methylpyridine (1.0 eq) was dissolved in toluene (12 vol). K₂CO₃ (4.8 eq) was added, followed by water (3.5 vol). The resulting mixture was heated to 65 ° C under a stream of n₂ for an hour

Acid

- (t-

<img file="AR080944A1_D0161.tif" />

Butoxycarbonyl) phenylboronic acid (1.05 eq) and Pd (dppf) Cl₂ CH₂Cl₂ (0.015 eq) were then added and the mixture was heated to 80 ° C. After 2 hours, the heat was turned off, water (3.5 vol) was added and the layers could be separated. The organic phase was then washed with water (3.5 vol) and extracted with 10% aqueous methanesulfonic acid (2 eq of MsOH, 7.7 voi). The aqueous phase was made alkaline with 50% aqueous NaOH (2 eq) and extracted with EtOAc (8 voi). The organic layer was concentrated to obtain 3— (3— methylpyridin-2-yl) crude tert-butyl benzoate (82%) which was used directly in the next step.

100172) Example 2j: 2- (3- (terButoxycarbon.yl) phenyl) -3-methylpyridine oxide

<img file="AR080944A1_D0162.tif" />

urea-hydrogen peroxide phthalic anhydride EtOAc, water

<img file="AR080944A1_D0163.tif" />

[00173) tert-Butyl 3- (3-methylpyridin-2-yl) benzoate (1.0 eq) was dissolved in EtOAc (6 voi). Water (0. 3 voi) was added, followed by urea-hydrogen peroxide (3 eq). Then, phthalic anhydride (3 eq) was added in portions to the mixture in the form of a solid at such a speed as to maintain the temperature in the reactor at less than 45 ° C. After completing the addition of phthalic anhydride, the mixture was heated to 45 ° C. After stirring for another 4 hours, the heat went out. Na₂SW₃ 10% aqueous w / w (1.5 eq) was added by means of addition funnel. After completing the addition of Na₂SW₃, the mixture was stirred for

<img file="AR080944A1_D0164.tif" />

another 30 min and the layers separated. The organic layer was shaken and Na was added₂CO₃ aqueous 10% w / w (2 eq). After stirring for 30 minutes, the layers could be separated. The organic phase was washed with 13% aqueous NaCl w / v. The organic phase was then filtered and concentrated to obtain crude 1— 2— (3 - (tert-butoxycarbonyl) phenyl) -3-methylpyridine oxide (95%) which was used directly in the next step.

[00174] Example 2k: tert-butyl 3- (6-amino-3-methylpyridin-2-yl) benzoate

<img file="AR080944A1_D0165.tif" />

one. Ms₂O. py. MeCN 70 degrees c

two. ethanolamine

<img file="AR080944A1_D0166.tif" />

100175] A solution of 2- (3- (terbutoxycarbonyl) phenyl) -3-methylpyridine (1 eq) and pyridine (4 eq) in acetonitrile (8 voi) was heated to 70 ° C. A solution of methanesulfonic anhydride (1.5 eq) in MeCN (2 voi) was added for 50 min by means of an addition funnel keeping the temperature below 75 ° C. The mixture was stirred for an additional 0.5 hours after completing the addition. The mixture was then allowed to cool to room temperature. Ethanolamine (10 eq) was added by means of addition funnel. After stirring for hours, water (6 voi) was added and the mixture was cooled to 10 ° C. After stirring for 3 hours, the solid was collected by filtration and washed with water (3 voi), 2: 1 acetonitrile / water (3 vol) and acetonitrile (2 x 1.5 vol). The solid dried to weight

<img file="AR080944A1_D0167.tif" />

constant (&lt;1% difference) in a vacuum homo at 50 ° C with a slight purge of N₂ to obtain tert-butyl 3- (6-amino-3-methylpyridin-2-yl) benzoate in the form of a reddish yellow solid (53% yield).

(001761 Example 21: 3- (6- (1- (2,2-Dif luorobenzo [d] [1,3] dioxol5-yl) -cyclopropancarboxamido) -3-methylpyridin-2-yl) -butylbenzoate.

<img file="AR080944A1_D0168.tif" />

<img file="AR080944A1_D0169.tif" />

[00177] The crude acid chloride described above was dissolved in toluene (2.5 vol based on acid chloride) and added by means of an addition funnel to a mixture of 3— (6-amino-3-methylpyridine -2-yl) tert-butyl benzoate (1 eq), DMAP, (0.02 eq) and triethylamine (3.0 eq) in toluene (4 vol based on 3 (6-amino-3-methylpyridin-2 -yl) tert-butyl benzoate). After hours, water (4 vol based on tert-butyl 3- (6-amino-3-methylpyridin-2-yl) benzoate) was added to the reaction mixture.

After stirring for 30 minutes, the layers separated. The organic phase was then filtered and concentrated to obtain a thick oil of 3- (6- (1- (2,2-difluorobenzo [d] [1,3] dioxol-5-yl) cyclopropancarboxamido) -3-methylpyridin- 2-yl) -t-butylbenzoate (quantitative crude yield). Acetonitrile (3 vol based on the crude product) was added and distilled until crystallization occurred. Water (2 vol based on crude product) was added

<img file="AR080944A1_D0170.tif" />

and the mixture was stirred for 2 h. The solid was collected by filtration, washed with 1: 1 (by volume) acetonitrile / water (2 x volumes based on the crude product) and partially dried in the vacuum filter. The solid was dried to a constant weight (&lt;1% difference) in a vacuum oven at 60 ° C with a slight purge of N₂ for difluorobenzo [d] [1,3] dioxol-5-yl) obtain 3- (6- (1- (2,2-cyclopropancarboxamido) -3-methylpyridin-2-yl) -t-butylbenzoate in the form of a brown solid.

[00178] Example 2m:

Difluorobenzo [d] [1,3] dioxol-5-yl) 3- (6- (1- (2,2-cyclopropancarboxamido) -3-methylpyridin-2-yl) benzoic acid · HCl sai

<img file="AR080944A1_D0171.tif" />

6N HCl MeCN

<img file="AR080944A1_D0172.tif" />

CO-H hc:

[00179] To a suspension of 3- (6- (1- (2,2difluorobenzo [d] [1,3] dioxol-5-yl) cyclopropancarboxamido) -3-methylpyridin-2-yl) -t-butylbenzoate (1.0 eq) in MeCN (3.0 voi) water (0.83 voi) was added followed by concentrated aqueous HCl (0.83 voi). The mixture was heated to 45 ± 5 ° C. After stirring for 24 to 48 h, the reaction was complete and the mixture was allowed to cool to room temperature. Water (1.33 voi) was added and the mixture was stirred. The solid was collected by filtration, washed with water (2 x 0.3 vol) and partially dried in the filter at

<img file="AR080944A1_D0173.tif" />

empty. The solid was dried to a constant weight (&lt;1% difference) in a vacuum oven at 60 ° C with a slight purge of

N₂ to obtain 3- (6- (1- (2,2-difluorobenzo [d] [1,3] dioxol-5il) cyclopropancarboxamido) -3-methylpyridin-2-yl) benzoic acid · HCI in the form of an off-white solid.

[00180] The following Table 2-1 indicates the physical data for compound 2.

Table 2-1,

<td></td><td>LC / MS</td><td colspan="4">LC / RT</td>

<td>Compound</td><td>M + 1</td><td>minutes</td><td>NMR</td><td></td><td></td>

<td></td><td></td><td></td><td>^ NMR (400</td><td>MHz, DMSO-d6)</td><td> 9 , 14</td>

<td></td><td></td><td></td><td>(YES H),</td><td>7.99-7.93 (m,</td><td>3H),</td>

<td></td><td></td><td></td><td> 7,80-7,78</td><td>(m, lH), 7.74</td><td> -7,72</td>

<td>Compound 2</td><td> 453,3</td><td> 1,93</td><td>(my h),</td><td>7.60-7.55 (m</td><td>, 2H),</td>

<td></td><td></td><td></td><td> 7,41-7,33</td><td>(m, 2H), 2.24</td><td>(s,</td>

<td></td><td></td><td></td><td>3H), 1.53-</td><td>1.51 (m, 2H),</td><td> 1,19-</td>

<td></td><td></td><td></td><td colspan="2">1.17 (m, 2H).</td><td></td>

II.C. The compounds of formula III

<img file="AR080944A1_D0174.tif" />

Rr

Formula III

<img file="AR080944A1_D0175.tif" />

II.Cl Embodiments of the compounds of the formula

III [00181] In one embodiment, in the compound of formula III:

R is H, OH, OCH₃ or two R taken together form -OCH₂Ou -OCF2O-;

<td colspan="5">R ^ is H 0 alkyl;</td>

<td>r₅</td><td>is</td><td>HO</td><td>F;</td><td></td>

<td>r₆</td><td>is</td><td>H 0</td><td>CN;</td><td></td>

<td>R7</td><td>is</td><td>H </td><td>-CH₂CH (OH) CH₂OH, -CH₂CH₂N⁺ (CH₃ ) ₃</td><td>0 -CH₂CH₂OH;</td>

<td>Rs</td><td>is</td><td>H</td><td>OH, -CH₂CH (OH) CH₂OH, -CH₂OH 0</td><td>Rv and R₈ take two</td>

together they form a ring of five members.

II.C.2. Compound 3

In another embodiment, the compound of the formula

III is compound 3, which is known by its chemical name (R) 1- (2,2-difluorobenzo [d] [1,3] dioxol-5-yl) -N- (1- (2,3-dihydroxypropyl) - 6-fluoro-2- (l-hydroxy-2-methylpropan-2-yl) -1 Hindol-5-yl) cyclopropancarboxamide.

<img file="AR080944A1_D0176.tif" />

compound 3

<img file="AR080944A1_D0177.tif" />

<img file="AR080944A1_D0178.tif" />

II.C.3. Summary of the synthesis of the compound [00182] Compound 3 can be prepared an acid chloride residue with a residue with the following schemes.

by amine coupling according

HC3.a. Synthesis of the acid residue of compound 3 [00183] The acid residue of compound 3 can be synthesized as acid chloride,

<img file="AR080944A1_D0179.tif" />

according to

Scheme 2-la, Scheme 2-lb and Examples 2a - 2h.

ll.C.3.b. Synthesis of the amine moiety of compound 3

Scheme 3-1: Synthesis of the rest of the amine.

<img file="AR080944A1_D0180.tif" />

<img file="AR080944A1_D0181.tif" />

[00184] Scheme 3-1 provides a summary of the synthesis of the amine moiety of compound 3. From the silyl protected propargilic shown, the conversion into the chloride of

<img file="AR080944A1_D0182.tif" />

Propargyl SvQ followed by Grignard reagent formation and subsequent nucleophilic substitution provides ((2,2-dimethylbut3-inyloxy) methyl) benzene, which is used at another stage of the synthesis. To complete the rest of the amine, first joke the

4- Nitro-3-fluoroaniline and then becomes the toluenesulfonic acid sai of (R) -1- (4-amino-2-bromo-5-fluorophenylamino) -3 (benzyloxy) propan-2-ol in one process two-stage starting with alkylation of the amino aniline group with (R) —2— (benzyloxymethyl) oxirane, followed by reduction of the nitro group in the corresponding amine. Palladium catalyzed coupling of the product with ((2,2-dimethylbut-3-inyloxy) methyl) benzene (previously treated) provides the alkynyl intermediate compound which is then cycled in the indole moiety to produce the amine moiety protected with Benzyl of compound 3.

II.C.3.C. Synthesis of compound 3 by coupling of the acid and amine moiety

Scheme 3-2. Formation of compound 3.

<img file="AR080944A1_D0183.tif" />

<img file="AR080944A1_D0184.tif" />

[00185) Scheme 3-2 represents the coupling of 1 acid and amine moieties to produce compound 3. In the first stage, (R) -l- (5-amino-2- (l- (benzyloxy) -2-methylpropan-2il) -6-fluoro-lH-indole-l-yl) -3- (benzyloxy) Propan-2-ol is coupled with 1- (2,2-difluorobenzo [d] [1,3] dioxol-5yl) cyclopropanecarbonyl chloride to provide the compound 3 protected with benzyl. This stage can be performed in the presence of a base and a solvent. The base can be an organic base like triethylamine and the solvent can be an organic solvent like DCM or a mixture of DCM and toluene.

[00186] In the last step, the benzylated intermediate is deprotected to produce compound 3. The deprotection step can be carried out using sufficient reduction conditions to remove the benzyl group. The reduction conditions may be hydrogenation conditions such as hydrogen gas in the presence of a palladium catalyst.

II.C.4. Examples: Synthesis of compound 3 ll.C.4.a. Compound 3 Synthesis of the rest of amine [00187]

Example 3a: 2-Bromo-5-fluoro-4-nitroaniline

ΛΛ

F NH₂

NBS

O? N

Br

EtOAc

50% [00188] A vessel was charged with 3-fluoro-4-nitroaniline (1.0 equiv) followed by ethyl acetate (10 vol) and stirred to dissolve all solids. N-Bromosuccinimide (1.0 equiv) is

<img file="AR080944A1_D0185.tif" />

added in portions to maintain an internal temperature of 2 ^SC. At the end of the reaction, the reaction mixture was concentrated in vacuo on a rotary evaporator. The residue was suspended in distilled water (5 voi) to dissolve and remove succinimide. (Succinimide can also be removed by water treatment.) The water was decanted and the solid was suspended in 2-propanol (5 voi) overnight. The resulting suspension was filtered and the wet cake was washed with 2-propanol, dried in the oven oven at 50^toC overnight with N purge₂ until constant weight. A yellowish roasted solid was isolated (50% yield, 97.5% AUC). Other impurities were a bromine regioisomers (1.4% of AUC) and a dibromo adduct (1.1% of AUC). ri NMR (500 MHz, DMSO) ò 8.19 (1 H, d, J =

8.1 Hz), 7.06 (br. S, 2 H), 6.64 (d, 1 H, J = 14.3 Hz).

[00189] Example 3b: Sai of p-toluenesulfonic acid of (K) -l ((4-amino-2-bromo-5-fluorophenyl) amino) -3- (benzyloxy) propan-2-ol.

OR₂N

F

<img file="AR080944A1_D0186.tif" />

Br

NH₂

1) cat, Zn (ClO,)₂-2H₂Or toluene. SO 'c 2) HyPt (S) C

IPAc

3) TsOH-H-, 0 DCM ©

<img file="AR080944A1_D0187.tif" />

[00190] A carefully dried container under N₂ It was charged with the following: 4 Â activated powder molecular sieves (50% by weight based on 2-bromo-5-fluoro-4-nitroaniline), 2Bromo-5-fluoro-4-nitroaniline (1.0 equiv), zinc perchlorate

<img file="AR080944A1_D0188.tif" />

dihydrate (20 mol%) and toluene (8 vol). The mixture was stirred at room temperature for no more than 3 0 min. Finally, (R) -bendi glycidyl ether (2.0 equiv) in toluene (2 vol) was added in a stable stream. The reaction was heated to 80²C (internal temperature) and stirred for approximately 7 hours or until 2-bromine 5 f luoro-4 nor trojan 1 ina was &lt;5% AUC.

[00191] The reaction was cooled to room temperature and

Celite® (50 p%) was added, followed by ethyl acetate (10 vol).

The resulting mixture was filtered to remove Celite® and sieves and washed with ethyl acetate (2 vol). The filtrate was washed with ammonium chloride solution (4 vol, 20% w / v). The organic layer was washed with sodium bicarbonate solution (4 vol x 2.5% w / v).

The organic layer was concentrated to the ford in a rotovap. The resulting suspension was dissolved in isopropyl acetate (10 vol) and this solution was transferred to a Buchi hydrogenator.

[00192] The hydrogenator was charged with 5% by weight of Pt (S) / C (1.5 mol%) and the mixture was stirred under N₂ at 30 ° C (internal temperature). The reaction was flooded with N₂ followed by hydrogen The pressure of the hydrogenator was adjusted to 1 bar of hydrogen and the mixture was rapidly stirred (&gt; 1200 rpm). At the end of the reaction, the catalyst was filtered through a plug of

Celite® and washed with dichloromethane (10 vol). The filtrate was concentrated in vacuo. All remaining isopropyl acetate was extracted with dichloromethane (2 vol) and concentrated on a rotary evaporator to dryness.

<img file="AR080944A1_D0189.tif" />

[00193] The resulting residue was dissolved in dichloromethane (10 voi). P-Toluenesulfonic acid monohydrate (1.2 equiv) was added and stirred overnight. The product was filtered and washed with dichloromethane (2 vol) and dried by suction. The wet cake was transferred to drying trays and in a vacuum oven and dried at 45^toC with N purge₂ until constant weight. The p-toluenesulfonic acid sai of (R) -l - ((4-amino-2-bromo-5 ~ fluorophenyl) amino) -3 - (benzyloxy) propan-2-ol was isolated as an off-white solid.

[00194]

Example 3c: (3-Chloro-3-methylbut-l-inyl) trimethylsilane

TMS

<img file="AR080944A1_D0190.tif" />

OH

Pure HCI

90%

TMS

<img file="AR080944A1_D0191.tif" />

CI [00195] Propargilic alcohol (1.0 equiv) is charged in a container. Aqueous hydrochloric acid (37%, 3.75 vol) was added and stirring began. During the dissolution of the solid alcohol, a modest endotherm was observed (5-6⁹C). The resulting mixture was stirred overnight (16 h), slowly turning dark red. A 30 L container with a shirt was charged with water (5 vol) which was then cooled to 10^andC. The reaction mixture was slowly transferred in water in vacuo, keeping the internal temperature of the mixture at less than 25 ^QC. Hexanes (3 vol) were added and the resulting mixture was stirred for 0.5 h. The phases were decanted and the aqueous phase (pH &lt;1) was drained and discarded. The organic phase was concentrated

<img file="AR080944A1_D0192.tif" />

in vacuo using a rotary evaporator, giving the product as a red oil.

[00196] 3d example: (4- (Benzyloxy) -3,3-dimethylbut-linyl) trimethylsilane.

TMS

<img file="AR080944A1_D0193.tif" />

Cl • Mg

two. BnOCH₂Cl _TMS

<img file="AR080944A1_D0194.tif" />

OBn

Method A [00197] All equivalents and volume descriptors in this part are based on a 250 g reaction. Magnesium chips (69.5 g, 2.86 moi, 2.0 equiv) were charged to a 3-L reactor with 4 mouths and stirred with a magnetic stirrer under nitrogen for 0.5 h. The reactor was immersed in an ice water bath. A solution of propargyl chloride (250 g, 1.43 moi, 1.0 equiv) in THF (1.8 L, 7.2 voi) was added slowly to the reactor, with stirring, until an initial exotherm was observed (approximately 10 ^toC). The formation of the Grignard reagent was conformed by IPC using N-NMR spectroscopy. Once the exotherm was over, the rest of the solution was added slowly, maintaining the temperature of the batch &lt;15 -C. The addition required approximately 3.5 h. The resulting dark green mixture was decanted in a capped 2 L bottle.

[00198] All the equivalents and volume descriptors in this part are based on a 500 g reaction. A 22 L reactor was charged with a solution of chloromethylbenzyl ether (95%, 375 g, 2.31 moi, 0.8 equiv) in THF (1.5 L, 3 vol). The reactor was cooled in an ice water bath. Two batches of reagent of i

<img file="AR080944A1_D0195.tif" />

slowly added to the chloromethylbenzyl ether solution by means of an addition funnel, keeping the batch temperature below 25 -C. The addition required 1.5 h. The reaction mixture was stirred overnight (16 h).

[00199J All equivalents and volume descriptors in this part are based on a 1 kg reaction. A solution of 15% ammonium chloride was prepared in a jacket reactor.

L (1.5 kg in 8.5 kg of water, 10 vol). The solution was cooled to 5^toC. Two Grignard reaction mixtures prepared as before were combined and then transferred to the ammonium chloride solution by means of an upper vessel. An exotherm was observed in this neutralization, which was carried out at a speed such as to keep the internal temperature below 25^SC. Upon completion of the transfer, the temperature of the vessel jacket was set at 25 ^QC. Hexanes (8 L, 8 vol) were added and the mixture was stirred for 0.5

h. After decanting the phases, the aqueous phase (pH 9) was drained and discarded. The remaining organic phase was washed with water (2 L, voi). The organic phase was concentrated in vacuo using a 22 L rotary evaporator, giving the crude product as an orange oil.

Method B [00200] Magnesium chips (106 g, 4.35 moi, 1.0 eq) were loaded into a 22 L reactor and then suspended in THF (7 60 mL, 1 vol). The vessel was cooled in a bath of ice water so that the batch temperature reached 2

<img file="AR080944A1_D0196.tif" />

<img file="AR080944A1_D0197.tif" />

of the propargyl chloride (760 g, 4.35 mol, 1.0 equiv) in THF (4.5 L, 6 vol) was added slowly to the reactor. After adding 100 mL, the addition was stopped and the mixture was stirred until an exotherm of 13 was observed.^SC, indicating the start of the Grignard reaction. Once the exotherm was over, another 500 mL of propargyl chloride solution was added slowly, keeping the batch temperature &lt;20^toC. Grignard reagent formation was confirmed with IPC using spectroscopy ¹H-NMR The rest of the propargyl chloride solution was added slowly, maintaining the temperature of the

<td>lot &lt;20</td><td>^toC.</td><td>The addition</td><td>required</td><td>approximately</td><td> 1,5</td><td>h.</td><td>The</td>

<td>solution</td><td>green</td><td colspan="3">resulting dark stirred during</td><td> 0,5</td><td>h.</td><td>The J i</td>

<td>training</td><td>of the</td><td>reagent of</td><td>Grignard</td><td>conformed by</td><td>CPI</td><td colspan="2">using! ì</td>

<td colspan="2">spectroscopy</td><td>'h-NMR. He</td><td colspan="3">cargo benzyl chloromethyl ether</td><td>pure</td><td>to i</td>

reactor addition funnel and then added dropwise to the reactor, keeping the batch temperature below 25 ^toC.

The addition required 1.0 h. The reaction mixture was stirred overnight. Aqueous work-up and concentration was carried out using the same procedure and relative amounts of materials as in Method A to give the product as an orange oil.

[00201] Example 3e: 4-Benzyloxy-3,3-dimethylbut-l-ino

TM S

Or Bn

Koh

MeOH

<img file="AR080944A1_D0198.tif" />

S 8% in 2 stages

OBn

<img file="AR080944A1_D0199.tif" />

[00202] A reactor with a 3 0 L jacket was charged with methanol vol) which was then cooled to 5 -C. Potassium Hydroxide (85%,

1.3 equiv) was added to the reactor. An exotherm of 1520 was observed²C when potassium hydroxide dissolved. The temperature of the shirt was set at 25^andC. A solution of 4-benzyloxy3,3-dimethyl-l-trimethylsilylbutyl-l-ino (1.0 equiv) in methanol (2 vol) was added and the resulting mixture was stirred until the reaction was completed, such as was controlled with HPLC Typical reaction time at 25²C was 3-4 h. The reaction mixture was diluted with water (8 vol) and then stirred for 0.5 h. Hexanes (6 vol) were added and the resulting mixture was stirred for 0.5 h.

The phases were allowed to decant and then the aqueous phase (pH 10-11) was drained and discarded. The organic phase was washed with a solution of KOH (85%, 0.4 equiv) in water (8 voi) followed by water (8 voi).

The organic phase was then concentrated using a rotary evaporator, resulting in the title material in the form of an orange-yellow oil. The typical purity of this material was in the range of 80% primarily with a single impurity present.^]H NMR (400 MHz, CéDè) δ 7.28 (d, 2 H, &lt;7 = 7.4

Hz), 7.18 (t, 2 H, 7 = 7.2 Hz), 7.10 (d, IH, 7 = 7.2 Hz), 4.35 (s, 2 H), 3.24 ( s, 2 H), 1.91 (s, 1 H), 1.25 (s, 6 H).

[00203] Example 3f: (R) -1- (4-amino-2- (4- (benzyloxy) -3,3dimethylbut-l-inyl) -5-fluorophenylamino) -3- (benzyloxy) propan-2-ol .

N. p Λ

<img file="AR080944A1_D0200.tif" />

<img file="AR080944A1_D0201.tif" />

... OBn

Pd (OAc) dppb K2CÒ2 Cul water

H₂N

F

<img file="AR080944A1_D0202.tif" />

Ύ 'OBn

OBn [00204] The (R) -1- (4-amino-2-bromo-5-fluorophenylamino) -3- (benzyloxy) propan-2-ol tosylate sai became the free base by stirring in dichloromethane (5 vol ) and saturated NaHCO solution₃ (5 vol) until a clear organic layer is achieved.

The resulting layers were separated and the organic layer was washed with saturated NaHCO solution.₃ (5 voi) followed by brine and concentrated in vacuo to obtain (R) -l- (4-amino-2-bromo-5fluorophenylamino) -3- (benzyloxy) propan-2-ol (free base) as a oil.

[00205] Palladium acetate (0.01 eq), dppb (0.015 eq), Cui (0.015 eq) and potassium carbonate (3 eq) were suspended in acetonitrile (1.2 voi). After stirring for 15 minutes, a solution of 4-benzyloxy-3,3-dimethylbut-l-ino (1.1 eq) in acetonitrile (0.2 voi) was added. The mixture was sprayed with nitrogen gas for 1 h and then a solution of (R) -l ((4-amino-2-bromo-5-f luorof enyl) amino) -3- (benzyloxy) propan-2- was added free base ol (1 eq) in acetonitrile (4.1 voi). The mixture was sprayed with nitrogen gas for another hour and then heated to 80 ^toC. The progress of the reaction was monitored with HPLC and the reaction was usually complete within 3-5 h. The mixture was cooled to room temperature and then filtered to

<img file="AR080944A1_D0203.tif" />

in Celite. The cake was washed with acetonitrile (4 vol).

Combined filtrates were azeotroped to dryness and then mixture was filtered in the next reactor. The acetonitrile solution of (R) -1 - ((4-amino-2- (4- (benzyloxy) -3,3-dimethylbut1-in-l-yl) -5-fluorophenyl) amino) -3- (benzyloxy) propan-2-ol thus obtained was used directly in the following procedure (cyclization) without further purification.

[00206] Example 3 g: (R) -1- (5-amino-2- (1- (benzyloxy) -2methylpropan-2-yl) -6-fluoro-lH-indole-l-yl) -3- ( benzyloxy) propan-2ol.

<img file="AR080944A1_D0204.tif" />

[00207] Bis-acetonitrilodichloropaladium (0.1 eq) and Cul (0.1 eq) were loaded into the reactor and then suspended in a solution of (R) -1- ((4-amino- 2- (4- (benzyloxy) -3,3-dimethylbut-lin-l-yl) -5-fluorophenyl) amino) -3- (benzyloxy) propan-2-ol obtained previously (1 eq) in acetonitrile (9.5 vol total) . The mixture was sprayed with nitrogen gas for 1 h and then heated to 80^PC. The progress of the reaction was monitored with HPLC and the reaction was typically complete within 1-3 h. The mixture was filtered through Celite and the cake was washed with acetonitrile. A solvent extraction was carried out in ethyl acetate (7.5 vol). The ethyl acetate solution was washed with aqueous NH3-NH4CI solution (2 x 2.5 vol) followed by 10%

<img file="AR080944A1_D0205.tif" />

brine (2.5 vol). The ethyl acetate solution was then stirred with silica gel (1.8 p eq) and Si-TMT (0.1 p eq) for 6

h. After filtering, the resulting solution was concentrated. The residual oil was dissolved in DCM / heptane (4 vol) and then purified by column chromatography. The oil obtained was then crystallized from 25% EtOAc / heptane (4 voi). (R) -I- (5-amino-2- (1- (benzyloxy) -2-methylpropan-2-yl) -6-fluoro-l Hindol-l-yl) -3- (benzyloxy) propan-2 was obtained -ol crystalline typically with a yield of 27-38%. NMR (400 MHz, DMSO) 7.38-7.34 (m, 4

<td>H), 7.32-7.23 (m,</td><td>6 H),</td><td> 7,21 (</td><td>d,</td><td>1 Η, J</td><td> = 12,8</td><td>Hz),</td><td>6.77 (d,</td><td>1 HOUR,</td>

<td>J = 9.0 Hz), 6.06</td><td>(s, 1</td><td>Η), 5,</td><td> 13</td><td>(d, IH,</td><td colspan="2">J = 4.9 Hz:</td><td> 1 , 4,54 (</td><td>s, 2</td>

<td>H), 4.46 (br. S,</td><td>2 Η),</td><td> 4,45</td><td>(s,</td><td>2 Η),</td><td> 4,33</td><td>(d, 1</td><td>H, J =</td><td> 12,4</td>

<td>Hz), 4.09-4.04 (m,</td><td>2 Η),</td><td> 3 , 63</td><td>(d,</td><td>IH, J</td><td> = 9,2</td><td>Hz),</td><td>3.56 (d,</td><td>1 HOUR,</td>

<td>J = 9.2 Hz), 3.49</td><td>(dd,</td><td>IH, J--</td><td> = 9,</td><td> ,8, 4,4</td><td>Hz),</td><td> 3,43</td><td>(dd, 1H,</td><td>J =</td>

<td>9.8, 5.7 Hz), 1.40</td><td>(s, 6</td><td>Η).</td><td></td><td></td><td></td><td></td><td></td><td></td>

II.C.4.b. Coupling [00208] Example 3h: Synthesis of (R) -N- (l- (3- (benzyloxy) -2-hydroxypropyl) -2- (1- (benzyloxy) -2-methylpropan-2-yl) -6-fluoro- lHindol-5-yl) -1- (2,2-difluorobenzo [d] [1,3] dioxol-5il) cyclopropancarboxamide.

<img file="AR080944A1_D0206.tif" />

EgX DCM, toluene

X ^F or

<img file="AR080944A1_D0207.tif" />

'ci

<img file="AR080944A1_D0208.tif" />

OBn

<img file="AR080944A1_D0209.tif" />

[00209J Acid 1- (2,2-Difluoro-1,3-benzodioxol-5-yl cyclopropancarboxylic acid (1.3 equiv) was suspended in toluene (2.5 voi, based on acid 1- (2,2- difluoro-1,3-benzodioxol-5-yl) cyclopropancarboxylic acid) Thionyl chloride (SOCI ?, 1.7 equiv) was added by means of an addition funnel and the mixture was heated to 60 ^fiC. The resulting mixture was stirred for 2 h.

Toluene and excess SOC1₂ They were distilled using a rotovap.

Additional toluene (2.5 vol, based on 1- (2,2-difluoro-1,3benzodioxol-5-yl) -cyclopropancarboxylic acid) was added and the mixture was distilled to 1 voi of toluene. A solution of (R) -1- (5 amino-2- (1- (benzyloxy) -2-methylpropan-2-yl) -6-fluoro-lH-indole-1yl) -3- (benzyloxy) propan-2- ol (1 eq) and triethylamine (3 eq) in DCM (4 voi) cooled to 0²C. The solution of acid chloride in toluene (1 voi) was added while maintaining the batch temperature below 10 ^QC. The progress of the reaction was monitored with HPLC and the reaction was usually complete in minutes. After heating up to 25^andC, the reaction mixture was washed with 5% NaHCCg (3.5 vol), 1 M NaOH (3.5 vol) and 1 M HC1 (5 voi). A solvent extraction in methanol (2 voi) was carried out and the resulting solution of (R) -N- (1- (3- (benzyloxy) -2-hydroxypropyl) -2- (1- (benzyloxy) -2-methylpropan -2-yl) -6-fluoro-lHindole-5-yl) -1- (2,2-difluorobenzo [d] [1,3] dioxo1-5il) cyclopropanecarboxamide in methanol was used without further purification in the next step ( hydrogenolysis).

[00210]

Example 3i: Synthesis of compound 3.

<img file="AR080944A1_D0210.tif" />

[00211] 5% palladium on carbon (-50% wet, 0.01 eq) was charged in an appropriate hydrogenation vessel. The solution of (R) -N- (1- (3 - (benzyloxy) -2-hydroxypropyl1) -2 - (1- (benzyloxy) -2methylpropan-2-yl) -6-fluoro-lH-indole-5- il) -1- (2,2difluorobenzo [d] [1,3] dioxol-5-yl) cyclopropanecarboxamide in methanol (2 vol) obtained above was carefully added, followed by a 3M solution of HCl in methanol. The vessel was purged with nitrogen gas and then with hydrogen gas. The mixture was vigorously stirred until the reaction was completed, as was determined by HPLC analysis. The typical reaction time was 3-5 h. The reaction mixture was filtered through Celite and the cake was washed with methanol (2 voi). A solvent extraction was performed in isopropanol (3 vol). The crude compound 3 was crystallized from 75% of IPA-heptane (4 vol, that is, 1 voi of heptane added to 3 vol of IPA) and the resulting crystals matured in 50% of IPA-heptane (i.e., 2 vol of heptane added to the mixture). Typical yields of compound 3 of the two-stage acylation / hydrogenolysis process ranges from 68% to 84%. Compound 3 can be recrystallized from IPAheptane according to the same procedure just described.

(00212] Compound 3 can also be prepared by means of one of the various synthetic routes disclosed in the application.

100

<img file="AR080944A1_D0211.tif" />

of published US patent, US 2009/0131492 preferably by reference.

incorporated into

<img file="AR080944A1_D0212.tif" />

Table 3-1: Physical data for compound 3.

<td>Comp.</td><td>LC / MS</td><td>LC / RT</td><td colspan="5">NMR</td>

<td>N. ²</td><td>M + l</td><td>min</td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td colspan="2">1 H NMR (400.0</td><td>MHz, CD₃CN) d</td><td> 7,69 (</td><td>d, J</td>

<td></td><td></td><td></td><td>= 7.7 Hz,</td><td>1 HOUR)</td><td>, 7.44 (d, J</td><td> = 1,6</td><td>Hz,</td>

<td></td><td></td><td></td><td>1H), 7.39 i</td><td>(dd</td><td>, J = 1.7, 8,</td><td>3 Hz,</td><td>1 HOUR) ,</td>

<td></td><td></td><td></td><td>7.31 (s, 1 H</td><td> ) ,</td><td>7.27 (d, J = 8</td><td>, 3 Hz,</td><td>1 HOUR) ,</td>

<td></td><td></td><td></td><td>7.20 (d, J</td><td> =</td><td>12.0 Hz, 1H)</td><td> , 6,34</td><td>(s,</td>

<td></td><td></td><td></td><td>1H), 4.32 (</td><td>d,</td><td colspan="2">J = 6.8 Hz, 2H), 4,</td><td> 15 -</td>

<td> 3</td><td> 521,5</td><td> 1,69</td><td colspan="2">4, 09 (m, 1 H),</td><td>3.89 (dd, J =</td><td> = 6,0,</td><td> 11,5</td>

Hz, 1H), 3.63-3.52 (m, 3H), 3.42 (d,

J = 4.6 Hz, 1H), 3.21 (dd, J = 6.2, 7.2

<td>Hz, 1H), 3.04 (t, J</td><td> = 5,8</td><td>Hz,</td><td>1 HOUR) ,</td><td> 1,59</td>

<td>(dd, J = 3.8, 6.8</td><td>Hz,</td><td>2H),</td><td> 1, 44</td><td>(s,</td>

<td>3H), 1.33 (s, 3H) and</td><td> 1,18</td><td>(dd,</td><td>J =</td><td> 3,7,</td>

<td>6.8 Hz, 2 H) ppm.</td><td></td><td></td><td></td><td></td>

III. FORMULATIONS [00213] In one aspect, the invention characterizes a formulation comprising a component selected from any embodiment described in Column A of the

Table I in combination with a component selected from any embodiment described in Column B and / or a

101 embodiment

<img file="AR080944A1_D0213.tif" />

component selected from any described in Column C of Table I.

[00214] Table I is reproduced here for convenience.

fai&gt;

<img file="AR080944A1_D0214.tif" />

Table I

<td>Shapes</td><td>from</td><td>Shapes</td><td colspan="2">from</td><td>Shapes</td><td>from</td>

<td>realization of</td><td>the</td><td>realization</td><td>from</td><td>the</td><td>realization</td><td>of the</td>

<td>Column A</td><td></td><td>Column B</td><td></td><td></td><td>Column C</td><td></td>

<td>Embodiments</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Section</td><td>Title</td><td>Section</td><td>Title</td><td>Section</td><td>Title</td>

<td>Il.A.1.</td><td>The</td><td>II.Bl</td><td>The</td><td>II.Cl</td><td>The</td>

<td></td><td>compounds</td><td></td><td>compounds</td><td></td><td>compounds</td>

<td></td><td>of the</td><td></td><td>of the</td><td></td><td>of the</td>

<td></td><td>formula I</td><td></td><td>formula II</td><td></td><td>formula</td>

<td></td><td></td><td></td><td></td><td></td><td>III</td>

<td>Il.A.2.</td><td>compound</td><td>II.B.2.</td><td>compound</td><td>II.C.2.</td><td>compound</td>

<td></td><td> 1</td><td></td><td> 2</td><td></td><td> 3</td>

100215] In an embodiment of this aspect, the formulation comprises an embodiment described in the

Column A in combination with an embodiment described in Column B. In another embodiment, the formulation comprises an embodiment described in Column A in combination with an embodiment described in Column

C. In another embodiment, the formulation comprises a combination of an embodiment described in the Column

<img file="AR080944A1_D0215.tif" />

102

<img file="AR080944A1_D0216.tif" />

A, an embodiment described in Column B and an embodiment described in Column C.

100216] In one embodiment of this aspect, the component of Column A is a compound of formula I. In another embodiment, the component of Column A is compound 1.

[00217] In one embodiment of this aspect, the component of Column B is a compound of formula II. In another embodiment, the component of Column B is compound 2.

[00218] In one embodiment of this aspect, the component of Column C is a compound of formula III. In another embodiment, the component of Column C is compound 3.

[00219] In one embodiment, the formulation comprises a homogeneous mixture comprising a composition according to Table I. In another embodiment, the formulation comprises a non-homogeneous mixture comprising a composition according to Table I.

[00220] The pharmaceutical composition of Table I can be administered in a vehicle or separately.

100221] In some embodiments, the pharmaceutical composition optionally comprises a pharmaceutically acceptable carrier, adjuvant or vehicle. In certain embodiments, these compositions optionally also comprise one or more additional therapeutic agents.

<img file="AR080944A1_D0217.tif" />

103 [00222] It will also be appreciated that some of the compounds of the present invention may exist in free form for treatment or, if necessary, as one of their pharmaceutically acceptable derivatives. In accordance with the present invention, a pharmaceutically acceptable derivative includes, but is not limited to, salts, esters, salts of these pharmaceutically acceptable esters or any adduct or derivative that, when administered to a patient in need, is able to provide, directly or indirectly, a compound as described herein elsewhere or one of its metabolites or residues.

[00223] Such as is used herein, the term "pharmaceutically acceptable sai" refers to salts that, within the scope of sound medical criteria, are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, and are associated with a reasonable risk / benefit ratio. A pharmaceutically acceptable sai implies any non-toxic sai or an ester sai of a compound of this invention which, when administered to a receptor, is capable of providing, either directly or indirectly, a compound of this invention or a metabolite or residue of it.

[00224] Pharmaceutically acceptable salts are well known in the art. For example, SM Berge et al. Describe pharmaceutically acceptable salts in detail in

<img file="AR080944A1_D0218.tif" />

104

J. Pharmaceutical Sciences, 1977, 66, 1-19, incorporate it into this reference. Pharmaceutically acceptable salts of the compounds of the present invention include those derived from suitable inorganic and organic acids and bases. Examples of salts by the addition of pharmaceutically acceptable non-toxic acids of an amino group formed with inorganic acids such as hydrochloric acid, hydrochloric acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxic acid, maleic acid , tartaric acid, citric acid, succinic acid or malonic acid or when using other methods used in the art as well as single exchange. Other pharmaceutically acceptable salts include salts of adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, canforate, caniorsulphonate, citrate, cyclopentanpropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, glutate sulfate, hemispherate, glutate, sulfate, glutate, sulfate, glutate, glutate, glutate, sulfate, glutate, sulfate, glutate, sulfate, glutate, sulfate, glutate, sulfate, glutate, sulfate, glutamate, glutate, sulfate, glutate, sulfate, glutate, sulfate, glutamate, glutate, sulfate, glutate, sulfate, phosphate , heptanoate, hexanoate, iohydrate, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, maionate, methanesulfonate, 2naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, ptoluensulfonate, undecanoate, and the like. The

<img file="AR080944A1_D0219.tif" />

105 salts derived from bases include alkali metal salts, alkaline earth metals, ammonium and N '(Ci alkyl.

₄)₄. The present invention also contemplates the quaternization of any nitrogen-containing basic group of the compounds described herein. Hydro- or fat-soluble or dispersible products can be obtained by said quaternization. Representative salts of alkali or alkaline earth metals include those of sodium, lithium, potassium, calcium, magnesium, and the like. Other pharmaceutically acceptable salts include, where appropriate, non-toxic cations ammonium, quaternary ammonium and amine formed with counterparts such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate and arylsulfonate.

[00225] Such a horn as described above, the pharmaceutically acceptable compositions of the present invention also comprise a pharmaceutically acceptable carrier, adjuvant or vehicle which, such horn as used herein, includes any and all solvents, diluents, or other vehicle. liquid, dispersion or suspension aid, surfactants, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as appropriate for the particular dosage form desired.

Remington's Pharmaceutical Sciences, 16^ç Edition, EW

Martin (Mack Publishing Co., Easton, Pa., 1980) describes

<img file="AR080944A1_D0220.tif" />

106 various carriers used in the formulation pharmaceutically acceptable compositions and known techniques for their preparation. Except when any conventional carrier medium is incompatible with the compounds of the invention, such as in producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component of the pharmaceutically acceptable composition, its use is contemplated within the scope of the present invention Some examples of materials that can serve as pharmaceutically acceptable carrier as include, without limitation, identical exchangers, alumina, aluminum stearate, lecithin, serum proteins, taies such as human serum albumin, phosphate buffers, glycine, sorbic acid, or sorbate. potassium, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, taies such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, polyacrylates, waxes, polyethylene block polypropylene polypropylene, lanolin, sugars such as lactose, glucose and sucrose; starches taies like 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;

<img file="AR080944A1_D0221.tif" />

107 excipients taies like cocoa butter and suppository waxes; Taies oils like peanut oil, cotton oil; Sassafras oil; Sesame oil; olive oil; corn oil and soy bean oil; glycols such as propylene glycol or polyethylene glycol; taies esters such as ethyl oleate and ethyl laureate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen free water; isotonic physiological solution; Ringer's solution; ethyl alcohol and phosphate buffer solutions, in addition to other compatible non-toxic lubricants such as sodium lauryl sulfate and magnesium stearate, in addition to coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants may also be present in the composition, according to the formulator's criteria.

[00226] The pharmaceutically acceptable compositions of the present invention can be administered to humans and other animals orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, typically (as powders, ointments or drops), bucai, as an oral spray. or nasal or similar, depending on the severity of the infection being treated. In certain embodiments, the compositions of the invention can be administered orally or parenterally at dosage levels of about 0.01 mg / kg to about 50 mg / kg and with

<img file="AR080944A1_D0222.tif" />

108 preference of about 1 mg / kg to about 2 mg / kg of the subject's body weight per day, once or several times per day, to obtain the desired therapeutic effect.

[00227] Liquid dosage forms for orai administration include, without limitation, emulsions, microemulsions, solutions, suspensions, syrups and pharmaceutically acceptable elixirs. In addition to the active compounds of the invention, 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 , ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3butylene glycol, dimethylformamide, oils (in particular, cotton semi-oil, peanut, corn, germ, olive, castor and sesame), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and esters of sorbitan fatty acids, and mixtures thereof. In addition to inert diluents, oral compositions may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents.

100228] Injectable preparations, for example, sterile aqueous or oily injectable suspensions may be formulated in accordance with known technique by suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can also be a solution,

<img file="AR080944A1_D0223.tif" />

109 suspension or sterile injectable emulsion in a nontoxic parenterally acceptable solvent diluent, for example, as a solution in 1,3-butanediol. Suitable vehicles and solvents that can be used include water, a solution of

Ringer, isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally employed as a solvent or suspension medium. For this purpose, any mild fixed oil, including mono or synthetic diglycerides, can be used. In addition, fatty acids, tais as oleic acid and their glyceride derivatives are useful in the preparation of injectables.

[00229] Injectable formulations can be sterilized, for example, by filtration through a filter that retains bacteria 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 before of its use

[00230] In order to prolong the effect of a compound of the present invention, it is often desirable to delay the absorption of the compound from the subcutaneous or intramuscular injection. This can be achieved by using a liquid suspension of crystalline or amorphous material with low water solubility. The rate of absorption of the compound then depends on its dissolution rate which in turn may depend on the size of the crystals and the crystalline form. Alternatively, by dissolving or suspending the compound in an oily vehicle,

<img file="AR080944A1_D0224.tif" />

110 achieves delayed absorption of a compound form administered parenterally. Injectable depot forms are prepared by forming microencapsulated matrices of the compound in biodegradable polymers such as polylactide polyglucolide. Depending on the ratio between compound and polymer, and the nature of the particular polymer used, the rate of release of the compound can be controlled. Examples of other biodegradable polymers include poly (orthoesters) and poly (anhydrides). Injectable depot formulations can also be prepared by crashing the compound into liposomes or microemulsions compatible with body tissues.

[00231] Compositions for rectal or vaginal administration preferably suppositories 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 is solid at room temperature, but liquid at rectal temperature and consequently melts in the rectum or vaginal cavity to release the active compound.

100232] Solid dosage forms for orai administration include capsules, tablets, pills, powders and granules. In said solid dosage forms, the active compound is mixed with at least one pharmaceutically acceptable inert carrier or excipient such as sodium citrate or dichloric phosphate and / or) fillers or extenders such as

<img file="AR080944A1_D0225.tif" />

Ili starches, lactose, sucrose, glucose, mannitol and silicone acid,

b) taies such as, for example, carboxymethyl cellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose and acacia,

c) taies corno glycerol humectants, d) taies corno agar-agar disintegrating agents, calcium carbonate, potato or tapioca starch, algic acid, certain silicates and sodium carbonate,

e) solution of taies corno paraffin retarding agents, f) taies corno absorption accelerators composed of quaternary ammonium, g) taies corno wetting agents for example, cetyl alcohol and glycerol monostearate, h) taies corno caolin absorbents and bentonite clay, and i ) Taies such as talcum, 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.

[00233] Solid compositions of a similar type may also be used as fillers in soft or hard filled gelatin capsules using excipients such as lactose or milk sugar, in addition to high molecular weight polyethylene glycols, and the like. The solid dosage forms of tablets, dragees, capsules, pills and granules can be prepared with covers and pods as well as enteric coated and other well-known covers in the pharmaceutical formulation technique. They may optionally contain opacifying agents and may also be a composition that releases the active ingredient (s) only or preferably, in

<img file="AR080944A1_D0226.tif" />

112 certain part of the intestinal tract, optionally in delayed form. Examples of inclusive compositions that can be used include polymeric substances and waxes. Solid compositions of a similar type can also be used as fillers in hard or soft gelatin capsules filled with excipients such as lactose or milk sugar in addition to high molecular weight polyethylene glycols, and the like.

[00234] The active compounds may also be in microencapsulated form with one or more excipients such as previously observed. The solid dosage forms of the tablets, dragees, capsules, pills and granules can be prepared with covers and envelopes such as enteric covers, controlled release covers and other covers well known in the pharmaceutical formulation technique. In said solid dosage forms, the active compound may 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, when compressing lubricants and other auxiliary tablets such as magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. They may optionally contain opacifying agents and may also be of a composition that releases only the active ingredient (s) or preferably, in a certain part of the intestinal tract,

113

<img file="AR080944A1_D0227.tif" />

optionally, in delayed form. Examples of inclusive compositions that can be used include polymeric substances and waxes.

[00235] Dosage forms for the typical or transdermal administration of a compound of the present invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches. The active component is mixed under sterile conditions with a pharmaceutically acceptable carrier and any necessary preservative or buffer, as needed. Also contemplated are ophthalmic formulations, ear drops and eye drops within the scope of the present invention. In addition, the present invention contemplates the use of transdermal patches, which have the additional advantage of providing controlled provision of a compound to the body. Said dosage forms may be prepared by dissolving or dispersing the compound in the appropriate medium. Absorption enhancers can also be used to increase the flow of the compound through the skin. Velocity can be controlled by providing a velocity control membrane or by dispersing the compound in a polymer matrix or gel.

[00236] It will also be appreciated that the compositions described herein can be administered concurrently, before or after one or more desired therapeutic or medical procedures. The particular combination of therapies (therapeutic agents or procedures) to

<img file="AR080944A1_D0228.tif" />

114 Use in a combined regimen will take into account the compatibility of the therapeutic agents and / or procedures desired 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, a compound of the invention can be administered concurrently with another agent used to treat the same disorder) or different effects can be achieved (e.g., 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 as appropriate for the disease or condition being treated.

100237] 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.

[00238] In one embodiment, the additional agent is an antibiotic. Examples of useful antibiotics herein include tobramycin, which includes tobramycin inhalation powder (TIP), azithromycin, aztreonam, which includes the aerosolized form of aztreonam, amikacin, which includes the liposomal formulations of these, ciprofloxacin, which includes formulations of this for administration by inhalation, levoflaxacin, which includes

<img file="AR080944A1_D0229.tif" />

115 its aerosolized formulations, and combinations of two antibiotics, for example, fosfomycin and tobramycin.

[00239] In another embodiment, the additional agent is mucolytic. Examples of useful mucolytics herein include Pulmozyme®.

[00240] In another embodiment, the additional agent is a bronchodilator. Examples of bronchodilators include albuterol, metaprotenerol sulfate, pyrbuterol acetate, salmeterol, or tetrabulin sulfate.

[00241] In another embodiment, the additional agent is effective in restoring the surface fluid of the airways of the lung. Such agents increase the movement of the sai inside and outside the cells, which allows the mucus in the pulmonary airway to hydrate more and, consequently, more easily purify. Examples of such agents include hypertonic saline solution, tetrasodium denufosol ([[(3S,

5R) -5- (4-amino-2-oxopyrimidin-l-yl) -3-hydroxyoxolan-2i 1] methoxy-hydroxy phosphoryl [[[(2R, 3 S, 4R, 5R) - 5 - (2, 4 dioxopyrimidin-l-yl) -3,4-dihydroxyoxolan-2-yl] methoxyhydroxyphi fori1] oxy-hydroxyphi fori1] hydrogen-fos fato), or bronchitol (inhalation mannitol formulation).

[00242] In another embodiment, the additional agent is an anti-inflammatory agent, that is, an agent that can reduce inflammation in the lungs. Examples of such useful agents herein include ibuprofen,

116

<img file="AR080944A1_D0230.tif" />

Docosahexanoic acid (DHA), sildenafil, inhalation glutati, pioglitazone, hydroxychloroquine or simvastatin.

[00243] In another embodiment, the additional agent is a different CFTR modulator of Compound 1, that is, an agent that has the effect of modulating the activity of

CFTR Examples of such agents include ataluren (PTC124®; 3- [5- (2-fluorophenyl) -1,2,4-oxadiazol-3yl] benzoic acid), sinapultide, lancovutide, depelestat (a recombinant human neutrophil elastase inhibitor ), cobiprostone (acid 7 - {(2R, 4aR, 5R, 7aR) -2 - [(3 S) -1,1di fluoro-3-methylpentyl] -2-hydroxy-6oxooctahydrocyclopenta [b] piran-5-i1} heptanoic), or acid (3 (6- (1- (2,2-difluorobenzo [d] [1,3] dioxol-5-i1)) cyclopropanecarboxamido) -3-methylpyridin-2-i1 (benzoic. In another embodiment, the additional agent is acid (3 (6- (1- (2,2-difluorobenzo [d] [1,3] dioxol-5-i1)) cyclopropanecarboxamido) -3-methylpyridin-2-i1) benzoic.

100244] In another embodiment, the additional agent is a nutritional agent. Examples of such agents include pancrelipase (pancreatic enzyme replacement), which includes Pancrease®, Pancreacarb®, Ultrase®, or Creon®,

Liprotomase® (formerly Trizytek®), Aquadeks®, or glutathione by inhalation. In one embodiment, the additional nutritional agent is pancrelipase.

100245] The amount of additional therapeutic agent present in the compositions of the present invention will not be greater

<img file="AR080944A1_D0231.tif" />

117 that the amount that would normally be administered in a composition comprising that therapeutic agent as a single active agent. Preferably the amount of additional therapeutic agent in the compositions described herein will vary from about 50% to 100% of the amount normally present in a composition comprising that single agent as therapeutically active agent.

[00246] A composition of the invention as described herein can also be incorporated into compositions for coating an implantable medical device, such as prostheses, artificial valves, vascular grafts, stents and catheters. According to it, the present invention, in another aspect, includes a composition for covering an implantable device comprising a compound of the present invention such as is described in general in advance and in classes and subclasses of the present and a suitable carrier for covering 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 described above in general and in classes and subclasses of the present and an appropriate carrier for covering said implantable device. Suitable covers and general preparation of suitable implantable devices are described in US Pat.^?

118

<img file="AR080944A1_D0232.tif" />

6.099.562 ;

5.886.026;

and 5,304,121.

The covers

<img file="AR080944A1_D0233.tif" />

These are generally biocompatible polymeric materials such as a polymer of hydrogel, polymethyldisiloxane, polycaprolactone, polyethylene glycol, polylactic acid, ethylene vinyl acetate, and mixtures thereof. The covers may optionally be coated by a suitable upper cover of fluorosilicone, polysaccharides, polyethylene glycol, phospholipids or combinations thereof to impart controlled release characteristics to the composition.

In order that the invention described herein may be more fully understood, the following examples are provided. It is to be understood that these examples are for illustrative purposes only and are not constructed as limiting of this invention in any way.

IV. METHODS OF USE [00247] In yet another aspect, the present invention provides a method of treating a pathological condition, disease or disorder involved by CFTR comprising a compound of the formula I in combination with a compound of the formula II and / or a compound of the formula III, which comprises the administration of the formulation to a subject, preferably a mammal, who needs it. In one embodiment, the pharmaceutical composition comprises compound 1 and compound 2. In another embodiment, the pharmaceutical composition comprises the

119 compound 1 and compound 3. In another form of pharmaceutical composition comprises the compound and compound 3. In another embodiment, pharmaceutical comprises components such as

Table I.

<img file="AR080944A1_D0234.tif" />

1, the compound the composition provided in [00248] In certain embodiments, the present invention provides a method of treating a pathological condition, disease or disorder involved by a deficiency of CFTR activity, which comprises the administration of the Pharmaceutical composition of the invention to a subject, preferably a mammal, who needs it.

[00249] In yet another aspect, the present invention provides a method of treatment or reduction of the severity of a pathological condition, disease or disorder involved by CFTR mutation. In certain embodiments, the present invention provides a method of treating a pathological condition, disease or disorder implicated by a deficiency of CFTR activity, which comprises the administration of the pharmaceutical composition of the invention to a subject, preferably a Mamifero, you need it.

[00250] In another aspect, the invention also provides a method of treating or reducing the severity of a disease in a patient, which comprises administering the pharmaceutical composition of the invention to a subject, preferably a mammal, who needs it. and said disease is selected from cystic fibrosis, asthma, COPD induced by

<img file="AR080944A1_D0235.tif" />

120 smoking, chronic bronchitis, rhinosinusitis, conspiration \ pancreatitis, pancreatic insufficiency, male infertility caused by congenital bilateral absence of the vas deferens (CBAVD), moderate pulmonary disease, idiopathic pancreatitis, allergic bronchopulmonary aspergillosis, hepatic hematism, liver disease hereditary, coagulation-fibrinolysis deficiencies, taies as protein C deficiency, hereditary type 1 angioedema, deficiencies in lipid processing, taies as familial hypercholesterolemia, type 1 chylomicronemia, abetalipoproteinemia, lysosomal storage diseases, taies as corneal I / pseudo-Hurler disease, mucopolysaccharidosis, Sandhof / TaiSachs, Crigler-Najjar type II, polyocrinopathy / hyperinsulemia , Diabetes mellitus, Laron's dwarfism, Mileoperoxidase deficiency, Primary hypoparathyroidism, Melanoma, Type 1 CDG glycanosis, Congenital hyperthyroidism, imperfect osteogenesis, hereditary hypofibrinogenemia, ACTa deficiency, diabetes insipidus (DI), neurophysal ID, neprogenic ID, Charcot-Marie Tooth syndrome, Perlizaeus-Merzbacher disease, neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis , progressive supranuclear paralysis, Pick's disease, various neurological disorders of polyglutamine taies like Huntington, spinocerebelar ataxia type I, spinal and bulbar muscular atrophy, palidoluisian and myotonic dentatorubal dystrophy, as well

121

<img file="AR080944A1_D0236.tif" />

as spongiform encephalopathies, taies as hereditary Creutzfeldt-Jakob disease (due to prion protein processing defect), Fabri disease, Straussler-Scheinker syndrome, COPD, dry eye disease or Sjogren's disease, osteoporosis, osteopenia, healing of bones and bone grop (including bone repair, bone regeneration, reduced bone resorption and increased bone deposition), Gorham syndrome, Chloeal taie corno myotonia congenital chloride (forms of Thomson and Becker), type III Bartter syndrome, Dent disease, epilepsy, hyperecplexia, lysosomal storage disease, Angelman syndrome and primary ciliary dysguinesia (PCD), a term for disorders inherited from the ciliary structure and / or function, including PCD with situs inversus (also known as a syndrome of

Kartagener), PCD without situs inversus and ciliary aplasia.

[00251] In some embodiments, the method includes treatment or reduction of the severity of cystic fibrosis in a patient that comprises administering to said patient one of the compositions as defined herein.

In certain embodiments, the patient possesses mutant forms of human CFTR. In other embodiments, the patient has one or more of the following AF508 mutations,

R117H and G551D of human CFTR. In one embodiment, the method includes treatment or reduction of the severity of cystic fibrosis in a patient who possesses the AF508 mutation of human CFTR comprising administration to said patient.

<img file="AR080944A1_D0237.tif" />

122 of one of the compositions as such is defined herein.

In one embodiment, the method includes treatment or reduction of the severity of cystic fibrosis in a patient who possesses the G551D mutation of human CFTR comprising the administration to said patient of one of the compositions as such is defined herein. . In one embodiment, the method includes treatment or reduction of the severity of cystic fibrosis in a patient who possesses the AF508 mutation of human CFTR in at least one allele comprising administering to said patient one of the compositions such as horn. It is defined herein. In one embodiment, the method includes treatment or reduction of the severity of cystic fibrosis in a patient who possesses the ÄF508 mutation of human CFTR in both alleles that comprises administering to said patient one of the compositions as defined. at the moment. In one embodiment, the method includes treatment or reduction of the severity of cystic fibrosis in a patient who possesses the G551D mutation of human CFTR in at least one allele comprising administering to said patient one of the compositions such as horn. It is defined herein. In one embodiment, the method includes treatment or reduction of the severity of cystic fibrosis in a patient who possesses the G551D mutation of human CFTR in both alleles comprising the administration to said patient of one of the compositions as defined. at the moment.

123

<img file="AR080944A1_D0238.tif" />

[00252] In some embodiments, the method includes reducing the severity of cystic fibrosis in a patient that comprises administering to said patient one of the compositions as such is defined herein. In certain embodiments, the patient possesses mutant forms of human CFTR. In other embodiments, the patient has one or more of the following AF508, R117H and G551D mutations of human CFTR. In one embodiment, the method includes reducing the severity of cystic fibrosis in a patient who possesses the AF508 mutation of human CFTR which comprises administering to said patient one of the compositions as defined herein. In one embodiment, the method includes reducing the severity of cystic fibrosis in a patient who possesses the G551D mutation of human CFTR which comprises administering to said patient one of the compositions as defined herein. In one embodiment, the method includes reducing the severity of cystic fibrosis in a patient who possesses the AF508 mutation of human CFTR in at least one allele comprising administering to said patient one of the compositions such as defined herein. In one embodiment, the method includes reducing the severity of cystic fibrosis in a patient who possesses the AF508 mutation of human CFTR in both alleles which comprises administering to said patient one of the compositions as defined in the present. In one embodiment, the method includes the

<img file="AR080944A1_D0239.tif" />

124 reduction of the severity of cystic fibrosis in a patient who possesses the G551D mutation of human CFTR in at least one allele comprising the administration to said patient of one of the compositions as such is defined herein. In one embodiment, the method includes reducing the severity of cystic fibrosis in a patient who possesses the G551D mutation of human CFTR in both alleles which comprises administering to said patient one of the compositions as such as defined in the present.

[00253] In some aspects, the invention provides a method of treating or reducing the severity of Osteoporosis in a patient comprising the administration to said patient of such a composition as defined herein.

[00254] In certain embodiments, the method of treatment or reduction of the severity of Osteoporosis in a patient comprises the administration to said patient of a pharmaceutical composition such as described herein.

[00255] In some aspects, the invention provides a method of treatment or reduction of the severity of osteopenia in a patient comprising the administration to said patient of such a composition as defined in the presence.

[00256] In certain embodiments, the method of treatment or reduction of the severity of osteopenia in a patient comprises the administration to said patient of a pharmaceutical composition such as described herein.

125 [00257] In some aspects, the invention provides a method of bone healing and / or bone repair in a patient comprising the administration to said patient of such a composition as defined herein.

[00258] In certain embodiments, the method of bone healing and / or bone repair in a patient comprises administering to said patient a pharmaceutical composition such as described herein.

[00259] In some aspects, the invention provides a method of reducing bone resorption in a patient that comprises administering to said patient a composition as defined herein, [00260] In some aspects, the invention provides a Method of increasing bone deposition in a patient comprising the administration to said patient of such a composition as defined herein.

[00261] In certain embodiments, the method of increasing bone deposition in a patient comprises the administration to said patient of such a composition as defined herein.

[00262] In some aspects, the invention provides a method of treating or reducing the severity of COPD in a patient that comprises administering to said patient a composition such as is defined herein.

100263] In certain embodiments, the method of treatment or reduction of the severity of COPD in a patient

<img file="AR080944A1_D0240.tif" />

126 it comprises the administration to said patient of such a composition as defined herein.

00264] In some aspects, the invention provides a method of treating or reducing the severity of COPD induced by smoking in a patient comprising the administration to said patient of such a composition as defined herein.

J00265] In certain embodiments, the method of treatment or reduction of the severity of COPD induced by smoking in a patient comprises the administration to said patient of such a composition as defined herein.

[00266] In some aspects, the invention provides a method of treatment or reduction of the severity of chronic bronchitis in a patient that comprises administering to said patient a composition such as described herein.

[00267] In certain embodiments, the method of treatment or reduction of the severity of chronic bronchitis in a patient comprises the administration to said patient of such a composition as defined herein.

[00268] According to an alternative embodiment, the present invention provides a method of cystic fibrosis treatment comprising the step of administering to said mammalian a composition such as is defined herein.

<img file="AR080944A1_D0241.tif" />

127 [00269] According to the invention, one of the composition is that amount &lt;

treatment or reduction of the severity of one or more of the diseases, disorders or pathological conditions, as previously stated.

[00270] Another aspect of the present invention provides a method of administration of a pharmaceutical composition by oral administration to a patient at least once a day of the composition as described herein. In one embodiment, the method comprises the administration of a composition to said patient of such a composition as defined herein once in Table I every 24 hours. In another embodiment, the method comprises the administration to said patient of such a composition as defined herein every 12 hours. In another embodiment, the method comprises the administration to said patient of such a composition as defined herein three times per day. In another embodiment, the method comprises the administration to said patient of such a composition as defined herein.

[00271] The compositions, according to the method of the present invention, can be administered using any amount and any route of effective administration for the treatment or reduction of the severity of one or more of the diseases, disorders or pathological conditions listed. at the moment.

128 [00272] In certain embodiments, the compositions

<img file="AR080944A1_D0242.tif" />

<img file="AR080944A1_D0243.tif" />

The present invention 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 and non-respiratory epithelia. The presence of residual CFTR activity on the epithelial surface can be easily detected using methods known in the art, for example, standard electrophysiological, biochemical or histochemical techniques. These methods identify the activity of CFTR using electrophysiological techniques in vivo or ex vivo, measurement of concentrations of Cl in perspiration and saliva, or biochemical or histochemical techniques ex vivo to control cell surface density. Using such methods, residual CFTR activity can be easily detected in heterozygous or homozygous patients with a variety of different mutations, which includes homozygous or heterozygous patients for the most common mutation, AF508.

[00273] In another embodiment, the compositions of the present invention are useful for the treatment or reduction of the severity of cystic fibrosis in patients who have an induced or increased residual CFTR activity using pharmacological methods or gene therapy.

These methods increase the amount of CFTR present on the cell surface, thus inducing a CFTR activity.

<img file="AR080944A1_D0244.tif" />

129 absent so far in a patient or increasing the existing level of residual CFTR activity in a patient.

[00274] In one embodiment, such a composition as defined herein may be useful for the treatment or reduction of the severity of cystic fibrosis in patients within certain genotypes exhibiting residual CFTR activity, for example. class mutations

III (altered regulation or blockage), class IV mutations (altered conductance) or class V mutations (reduced synthesis) (Lee R. Choo-Kang, Pamela L., Zeitlin, Type I, li,

III, IV, and V cystic fibrosis Tansmembrane Conductance

Regulator Defects and Opportunities of Therapy; Current

Opinion in Pulmonary Medicine 6: 521-529, 2000). Other patient genotypes that exhibit a residual CFTR activity include homozygous patients for one of these classes or heterozygous for any other class of mutations, including class I mutations, class II mutations or a mutation that lacks classification.

100275] In one aspect, the invention includes a method of treating a class III mutation such as described above, which comprises administration to a patient in need thereof a composition comprising a compound of the formula I in combination with one or the two compounds of the formula II and / or compounds of the formula III. In some embodiments of this aspect, the composition includes a compound of the formula I in combination with a compound of the

<img file="AR080944A1_D0245.tif" />

130 Formula II In some embodiments of this aspect, 1 'composition includes a compound of the formula I in combination with a compound of the formula III. In some embodiments of this aspect, the composition includes a compound of the formula I in combination with a compound of the formula II and a compound of the formula III. In another embodiment of this aspect, the pharmaceutical composition includes compound 1 and compound 2. In another embodiment, the pharmaceutical composition includes compound 1 and compound

3. In another embodiment, the pharmaceutical composition includes compound 1, compound 2 and compound 3.

[00276] In one embodiment, such a composition as defined herein may be useful for the treatment or reduction of the severity of cystic fibrosis in patients within certain clinical phenotypes, for example, a moderate clinical phenotype. to mild that typically correlates with the amount of activity of

CFTR residuai 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 the vas deferens or moderate lung disease.

[00277] 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 compositions of

<img file="AR080944A1_D0246.tif" />

131 The invention is preferably formulated in a dosage unit form to facilitate administration and standardize the dosage. The term "dosage unit form" as used herein refers to a physically discrete unit of 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 treating physician within the scope of good medical judgment. The specific effective dose level for any particular patient or organism will depend on a variety of factors including the treatment disorder and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general status, 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 treatment; the drugs used in combination or coinciding with the specific compound employed and related factors well known in medical techniques. The term "patient", as used herein, implies an animal, preferably a mammal and, most preferably, a human being.

[00278] In one aspect, the present invention characterizes a kit comprising such a composition as defined herein.

<img file="AR080944A1_D0247.tif" />

132

V. TESTS

VA Protocol 1 [00279] Assays to detect and measure the potentiation properties of AF508-CFTR of the compounds

Optical membrane potential methods to test the modulation properties of hF508-CFTR of compounds [00280] The assay uses fluorescent voltage sensing matrices to measure changes in membrane potential using a fluorescent plate reader (eg FLIPR III , Molecular Devices, Ine.) As a reading for the increase of functional AF508-CFTR in cells

NIH 3T3. The driving force for the response is the creation of a chloride ion gradient along with the activation of the channel by a simple liquid addition step after having previously treated the cells with compounds and subsequently loaded with a voltage sensing matrix.

Identification of enhancer compounds [00281] To identify the enhancers of AF508-CFTR, a double-addition HTS assay format was developed.

This HTS assay uses fluorescent voltage sensing matrices to measure changes in membrane potential in FLIPR III as measured for the increase in conductance of AF508 CFTR in AF508 CFTR NIH 3T3 cells with corrected temperature. The driving force for the response is a gradient of Cl 'ion along with the activation

<img file="AR080944A1_D0248.tif" />

133 of the channel with forskolin in a simple liquid addition stage using a FLIPR III fluorescent plate reader after having previously treated the cells with enhancer compounds (or control with DMSO vehicle) and subsequently loaded with a redistribution matrix.

Solutions [00282]

Bath solution # 1: (in mM) NaCI 160, KCl 4,5,

CaCl₂ 2, MgCl₂ 1, HEPES 10, pH 7.4 with NaOH.

[00283]

CHLORINE-FREE BATH SOLUTION: THE SALTS OF

CHLORINE IN BATH SOLUTION # 1 REPLACE WITH SALTS OF

GLUCONATE

Cell culture [001] NIH3T3 mouse fibroblasts stably expressing AF508-CFTR are used for optical measurements of membrane potential. The cells are maintained at 37 ° C in

5% CO₂ and 90% humidity in Eagle medium modified with

Dulbecco supplemented with 2 mM glutamine, 10% fetal bovine serum, 1 X NEAA, ß-ME, 1 X pen / estrep and 25 mM HEPES in 175 cm culture vessel². For all optical tests, the cells were seeded at ~ 20,000 / cavity in 384 cavity coated matrices and grown for 2 h at 37^QC before growing to 27 ²C for 24 h for the enhancer test. For correction assays, cells are grown at 27²C or 37

<img file="AR080944A1_D0249.tif" />

134 ^SC with and without compounds for 16-24 hours. Electrophysiological assays were used to test the modulation properties of AF508-CFTR of the compounds.

Testing with Ussing camera [00285] With the Ussing camera, experiments were conducted on epithelial cells of polarized airways expressing AF508-CFTR to then characterize AF508-CFTR modulators identified in optical tests. The epithelia of the non-CF and CF airways of bronchial tissue were isolated, cultured as previously described (Galletta, LJV, Lantero, S.,

Gazzolo, A., Sacco, 0., Romano, L., Rossi, GA, &amp; ZegarraMoran, 0. (1998) In Vitro Celi. Dev. Biol. 34, 478-481) and placed in Costar® Snapwell filters^IM which were prerecovered with conditioned medium with NIH3T3. After four days, the apical media were removed and the cells were grown in a liquid and air interface&gt; 14 days before using. This resulted in a monolayer of completely differentiated column cells that ciliated, characteristic of airway epithelia.

Non-CF HBE were isolated from non-smokers who did not have lung disease. CF-HBE were isolated from homozygous patients for AF508-CFTR.

[00286] HBE cultured in Costar® Snapwell ™ cell culture inserts were mounted in a Ussing chamber (Physiologie

Instruments, Inc., San Diego, CA) and the

135

<img file="AR080944A1_D0250.tif" />

in the presence of a graceful Dasoiaterai t (I_SC) using a voltage shutdown system (Department of Bioengineering, University of Iowa, IA). Briefly, HBE were examined under conditions of voltage clamp registration (V_mant = 0 mV) at 37 ° C. The basolateral solution contained (in mM) 145 NaCl, 0.83 K2HPO4, 3.3 KH2PO4,

1.2 MgCl₂, 1.2 CaCl₂, 10 Glucose, 10 HEPES (pH adjusted to 7.35 with NaOH) and the apical solution contained (in mM) 145 Na gluconate, 1.2 MgCl₂, 1.2 CaCl₂, 10 glucose, 10 HEPES (pH adjusted to 7.35 with NaOH).

Identification of enhancer compounds [00287] The typical protocol used a concentration gradient of Cl from basolateral to apical membrane. To fix this gradient, normal rings were used in the basolateral membrane, while the apical NaCl was replaced by equimolar sodium gluconate (titrated to pH

7.4 with NaOH) to give a large concentration gradient of

Cl through the epithelium. Forskolin (10 μΜ) and all test compounds were added to the apical side of the cell culture inserts. The efficacy of the putative enhancers of AF508-CFTR was compared with that of the known enhancer, genistein.

Patch-clamp records [00288] Total Cl current in cells was controlled

AF508-NIH3T3 using patch log configuration

<img file="AR080944A1_D0251.tif" />

136 perforated such horn as previously described (Rae, J .1,

Cooper, K., Gates, P., &amp; Watsky, M. (1991) J. Neurosci.

Methods 37, 15-26). Voltage clamp registration was performed at 22^toC using a patch-clamp amplifier

Axopatch 200B (Axon Instruments Inc., Foster City, CA). The pipette solution contained (in mM) 150 M-methyl-nglucamine (NMDG) -Cl, 2 MgCl₂, 2 CaCl₂, 10 of EGTA, 10 of HEPES and 240 pg / ml of amphotericin-B (pH adjusted to 7.35 with HCl). The extracellular medium contained (in mM) 150 of

NMDG-C1, 2 MgCl₂, 2 CaCl₂, 10 HEPES (pH adjusted to

7.35 with HCI). Pulse generation, data acquisition and analysis were performed using a PC equipped with a Digidata 1320 A / D interface together with Clampex 8 (Axon

Instruments Ine.). To activate ÄF508-CFTR, 10 μΜ of forskolin and 20 μΜ of genistein were added to the barium and the current-voltage ratio was checked every 30 sec.

Identification of enhancer compounds [002891 The capacity of enhancers of AF508-CFTR to increase Cl current was also investigated^- Macroscopic AF508CFTR (I _Ρ508 ) in NIH3T3 cells stably expressing AF508-CFTR using perforated patch registration techniques. The enhancers identified in the optical assays evoked a dose-dependent Ιήκ ^ β increase with similar potency and an efficacy observed in the optical assays. In all the cells examined, the potential

<img file="AR080944A1_D0252.tif" />

inverse before and during approximately -30 mV, which

137 Enhancer application was is the calculated ECi (-28 mV).

<img file="AR080944A1_D0253.tif" />

Cell culture

100290] NIH3T3 murine fibroblasts stably expressing AF508-CFTR are used for whole cell records.

The cells are kept at 37 ^SC in 5% CO₂ and 90% humidity in Dulbecco-modified Eagle medium supplemented with 2 mM glutamine, 10% fetal bovine serum, 1 X NEAA, ß-ME, 1 X pen / estrep and 25 mM HEPES in 175 cm culture bottles². For the whole cell records, 2,500-5,000 cells were seeded in glass coverslips coated with poly-L-lysine and cultured for 24 48 h at 27 -C before using to assay the activity of the enhancers; and incubated with or without the correction compound at 37^toC to measure the activity of the correctors.

Single channel records [00291] The blocking activity of wt-CFTR and AF508-CFTR with corrected tempo in NIH3T3 cells was observed using inside-out membrane patch records as described above (Dalemans, W., Barbry ,

P., Champigny, G., Jallat, S., Dott, K., Dreyer, D.,

Crystal, RG, Pavirani, A., Lecocq, JP., Lazdunski, M.

(1991) Nature 354, 526-528) using an Axopatch 200B patch-clamp amplifier (Axon Instruments Inc.). Pipette

138

<img file="AR080944A1_D0254.tif" />

contained (in mM): 150 of NMDG, 150 of aspartic acid, 5 of

CaCl₂, 2 MgCl₂ and 10 HEPES (pH adjusted to 7.35 based

Tris). The bath contained (in mM): 150 NMDG-C1, 2 MgCl₂, from EGTA, 10 from TES and 14 from Tris base (pH adjusted to 7.35 with HCl). After cutting, both wt-corno AF508-CFTR were activated by adding 1 mM Mg-ATP, 75 nM of the catalytic subunit of the cAMP-dependent protein kinase (PKA;

Promise Corp. Madison, WI) and 10 mM NaF to inhibit protein phosphatases, which prevent current flow. The pipette potential was maintained at 80 mV. Channel activity was analyzed from membrane patches with 2 active channels. The maximum number of simultaneous openings determined the amount of active channels during the course of an experiment. To determine the amplitude of the single channel current, the recorded data of 120 seconds of AF508-CFTR activity was filtered off-line at 100 Hz and then used to construct the amplitude histograms at all points of the construct that were adjusted with multigaussian functions using Bio-Patch software

Analysis (Bio-Logic Comp. France). The total microscopic current and the probability of opening (P_or) were determined from 120 seconds of channel activity. The P_or was determined using the Bio-Patch software or the relationship P_or =

I / i (N), where I = means current, i = amplitude of single channel current and N = number of active channels in patch.

139

<img file="AR080944A1_D0255.tif" />

Cell Culivo&gt;

«

<img file="AR080944A1_D0256.tif" />

[00292] NIH3T3 murine fibroblasts stably expressing AF508-CFTR are used for cut-membrane patch-clamp records. The cells are kept at 37^ÇC in 5% of

CO₂ and 90% humidity in Dulbecco-modified Eagle medium supplemented with 2 mM glutamine, 10% fetal bovine serum, 1 X NEAA, ß-ME, 1 X pen / estrep and 25 mM HEPES in 175 cm culture vessel². For single channel records, 2,500-5,000 cells were seeded in glass coverslips coated with poly-L-lysine and cultured during

- 48 to 27 ^toC before using.

Examples: Activity of the compounds of the formula I [00293] The compounds of the formula I are useful as modulators of the transporters of the ATP binding cassette.

Examples of activities and efficiencies of the compounds of formula I are indicated below in Table 1-2. The activity of the compound is illustrated with +++ when the measured activity was less than 2.0 μΜ, ++ when the measured activity was 2 μΜ a

5.0 μΜ, when the measured activity was greater than 5.0 μΜ and when there was no data available. Efficacy is illustrated with + + + when the efficacy was greater than 100%, + + when the efficacy was 100% to 25%, + when the efficacy was less than 25% and when there were no data available. It should be noted that 100% efficiency is the maximum response obtained with 4-methyl-2- (5-phenyl-1H-pyrazol-3-yl) phenol.

<img file="AR080944A1_D0257.tif" />

140 % efficiency

Table 1-2

Activities and efficiencies of the compounds of formula I

Activity

EC₅₀ (p.m)

Example compound N.

1-2

1-3

1-4

1-5

1-6

1-7

1-8

1-9

1-10

1-11

1-12

1-13

1-14

VC Protocol 2 [00294] Tests to detect and measure the correction properties of AF508-CFTR of the compounds [00295] Optical methods of membrane potential to test the modulation properties of AF508-CFTR of compounds

<img file="AR080944A1_D0258.tif" />

<img file="AR080944A1_D0259.tif" />

141 [00296] The membrane potential optical test utilizes voltage-sensitive FRET sensors described by Gonzalez and Tsien (See_± Gonzalez, JE and RY Tsien (1995) Voltage sensing by fluorescence résonance energy transfer in single cells Biophys J 69 (4): 1272-80, and Gonzalez, JE and RY

Tsien (1997) Improved indicators of celi membrane potential that use fluorescence résonance energy transfer Chem Biol

4 (4): 269-77) in combination with the instrumentation for measuring fluorescence changes taies as the reader

Ionic Voltage / Probe (VIPR) (See, Gonzalez, JE, K. Oades, et al. (1999) Cell-based assays and instrumentation for screening ion-channel targets Drug Discov Today 4 (9): 431439).

[00297] These voltage-sensitive assays are based on the change of fluorescence resonant energy transfer (FRET) between the membrane-sensitive, voltage-sensitive matrix, DiSBAC₂(3) and a fluorescent phospholipid, CC2-DMPE, which is attached to the leaflet of the piasmatic membrane and acts as a FRET donor. Changes in membrane potential (V_{: n}) cause the

DiSBAC₂(3) with a negative charge to redistribute through the piasmatic membrane and the amount of the energy transfer of CC2-DMPE changes accordingly. Changes in fluorescence emission can be controlled using VIPR¹"II, which is an integrated liquid controller and fluorescent detector designed to conduct sieves in

<img file="AR080944A1_D0260.tif" />

142 cavities

Identification of correction compounds

100298] To identify small molecules that correct the circulation defect associated with / _ \ F508-CFTR; a simple addition HTS assay format was developed. The cells were incubated in a serum-free medium for 16 hours at 37 ° C in the presence or absence (negative control) of the test compound. As a positive control, the cells were incubated in 384-well plates for 16 hours at 27 ° C with AF508-CFTR of the correct temperature. The cells were subsequently washed 3X with Krebs Ringer's solution and loaded with voltage sensitive dyes. To activate / AF508-CFTR, 10 μΜ of forskolin and the CFTR enhancer, genistein (20 μΜ) were added, together with the Cl ~ free medium to each well. The addition of the free medium of

Cl promoted the exit of Cl in response to the activation of

AF508-CFTR and the resulting membrane depolarization was optically controlled using voltage-sensitive dyes based on FRET.

Identification of enhancer compounds [00299] To identify enhancers of AF508-CFTR, a double-added HTS assay format was developed.

During the first addition, Cl-free medium with or without test compound was added to each well. After 22 seconds, a second addition of free medium was added

143

<img file="AR080944A1_D0261.tif" />

CFTR The extracellular concentration of additions was 28 mM, which promoted response to the activation of AF508-CFTR and the resulting membrane depolarization was optically controlled using voltage-sensitive dyes based on FRET.

Solutions (00300) Solution of bath # 1: (in mM) 160 of NaCI, 4.5 of KC1, 2 of CaCl₂, 1 MgCl₂, 10 HEPES, pH 7.4 with NaOH.

[00301] Chlorine-free bath solution: The chloride salts of the # 1 bath solution are substituted with gluconate salts.

[00302] CC2-DMPE: A standard solution of 10 mM in DMSO is prepared and stored at -20 ° C.

[00303] DiSBAC2 (3): A standard solution of mM in DMSO is prepared and stored at -20 ° C.

Cell culture [00304] NIH3T3 mouse fibroblasts that are stably expressing AF508-CFTR are used for optical measurements of membrane potential. The cells are kept at 37^BC in

5% CO₂ and 90% humidity in modified Eagle medium of

Dulbecco supplemented with 2 mM glutamine, 10% fetal bovine serum, 1 X NEAA, ß-ME, 1 X pen / estrep, and 25 mM of

HEPES in 175 cm culture flasks². In all optical tests, cells were seeded at the rate of

30,000 / well in plates coated with gel matrix

<img file="AR080944A1_D0262.tif" />

144

384 wells and grown for 2 hours at 37 ° C before growing at 27 ° C for 24 hours for the enhancer assay. In the correction assays, the cells are cultured at 27 ° C or 37 ° C with and without compounds for 16-24 hours.

[00305] Electrophysiological tests to examine the modulation properties of AF508-CFTR of the compounds

Ussing Chamber Assay [00306] Ussing chamber experiments were performed on polarized epithelial cells expressing / AF508-CFTR to further characterize the AF508-CFTR modulators identified in the optical assays. FRT epithelial cells^{F 508}~^CFTR grown in Costar Snapwell cell culture inserts were mounted in a chamber of

Ussing (Physiologie Instruments, Ine., San Diego, CA), and the monolayers were short-circuited continuously using a voltage clamp system (Department of

Bioengineering, University of Iowa, IA, and, Physiologie

Instruments, Inc., San Diego, CA). The transepiotential resistance was measured by the application of a 2 mV pulse. Under these conditions, the FRT epithelia showed resistance of 4 Ω / cm² or more. The solutions were kept at 27^andC and bubbled with air. The contact potential of the electrode and the resistance of the fluid were corrected using a cell-free insert.

Under these conditions, the current reflects the flow of Cl a

145

<img file="AR080944A1_D0263.tif" />

digitally obtained I_SC using an MPlOOA-CE interface and AcqKnowledge computer program (v3, 2, 6; BIOPAC

Systems, Santa Barbara, CA).

Identification of correction compounds [00307] The typical protocol used a Cl ”concentration gradient from basolateral to apical membrane. To establish this gradient, a normal Ringer solution in the basolateral membrane was used, while the apical NaCI was replaced with equimolar sodium gluconate (titrated at pH 7.4 with NaOH) to give a large Cl ”concentration gradient to through the epithelium All experiments were performed with intact monolayers. For total activation of AF508-CFTR, forskolin (10 pM) and the PDE inhibitor, IBMX (100 μΜ) were applied, followed by the addition of the CFTR enhancer, genistein (50 pM).

100308] As observed in other cell types, incubation at low temperatures of FRT cells stably expressing AF508-CFTR increases the functional density of CFTR in the piasmatic membrane. To determine the activity of the correction compounds, the cells were incubated with 10 pM of the test compound for 24 hours at 37 ° C and subsequently washed 3X before registration. The IF_SC-mediated cAMP and genistein in cells treated with the compound normalized in the controls at 27

C and 37 ° C and were expressed as a percentage of activity. The

146

<img file="AR080944A1_D0264.tif" />

preincubation of the cells with the correction compound significantly increased the I_SC mediated cAMP and genistein compared to 37 ° C controls.

Identification of enhancer compounds [00309} The typical protocol used a concentration gradient of Cl from the basolateral to apical membrane.

To establish this gradient, a solution of

Normal Ringer in the basolateral membrane and permeabilized with nystatin (360 pg / ml), while the apical NaCl was replaced with equimolar sodium gluconate (titrated to pH

7.4 with NaOH) to give a large concentration gradient of Cl through the epithelium. All experiments are

<td>performed</td><td> 30</td><td>minutes later</td><td>of the</td><td colspan="3">permeabilization</td><td>with</td>

<td>Nystatin</td><td>He</td><td colspan="2">they added forskolin ι</td><td> (10</td><td>μΜ)</td><td>and everyone</td><td>the</td>

<td>compounds</td><td>from</td><td>essay to both</td><td>sides</td><td>from</td><td>the</td><td>inserts</td><td>of the</td>

cell culture. The efficacy of the putative AF508-CFTR enhancers was compared with that of the known enhancer, genistein.

Solutions

100310) Basolateral solution (in mM): NaCl (135), CaCl₂ (1,2), MgCl₂ (1,2), K₂HPO₄ (2,4), KHPO₄ (0.6), N-2-hydroxyethylpiperazin-N'-2-ethanesulfonic acid (HEPES) (10), and dextrose (10). The solution was titrated to pH 7.4 with N aOH.

[00311] Apical solution (in mM): Same as basolateral solution with NaCl replaced with Na gluconate (135).

147

<img file="AR080944A1_D0265.tif" />

Fisher rat epithelial cells (FRT were used

Cell culture (00312) expressing AF508-CFTR (FRT

Z \ F5 0 8 ~ CFTR for the Ussing chamber experiments for the putative modulators / AF508CFTR identified in our optical tests. Cells were cultured in Costar Snapwell cell culture inserts and cultured for five days at 37 ° C and 5% C0₂ in F-12 modified Ham Coon medium supplemented with 5% fetal sheep serum, 100 U / ml penicillin and 100 pg / ml streptomycin. Before using for the characterization of 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 correction activity of the compounds, the cells were incubated at 27 ° C or 37 ° C with and without the compounds for 24 hours.

Full cell registration

100313] The current of macroscopic AF508-CFTR (I_toe5os) in NIH3T3 cells stably expressing temperature-corrected AF508-CFTR and test compounds was monitored using the full cell registry with perforated patch. In short, the voltage clamp registers of Ι_Λγ ·· 508 were performed at room temperature using an Axopatch 200B zonal clamp amplifier (Axon Instruments Ine., Foster City, CA). All records were obtained with a sampling frequency of 10 kHz and a low-pass filter at 1 kHz. The pipettes presented

148

<img file="AR080944A1_D0266.tif" />

a resistance of 5-6 Ω when loaded with the intracellular solution. Under these recording conditions, the investment potential calculated for Cl ~ (ECJ at room temperature was -28 mV. All records presented a sealing resistance&gt; 20 ΟΩ and a series resistance &lt;15 ΜΩ. Pulse generation, obtaining of data and analysis were performed using a PC equipped with a Digidata interface

1320 A / D in conjunction with Clampex 8 (Axon Instruments Ine.).

The batch contained &lt;250 μΐ of saline solution and was continuously pre-fused at a rate of 2 ml / min using a gravity-operated perfusion system.

Identification of correction compounds

100314] To determine the activity of correction compounds to increase the density of functional AF508-CFTR in the piasmatic membrane, we use the perforated patch recording techniques described above to measure the current density after hours of treatment with the compounds of correction.

To fully activate AF508-CFTR, 10 μΜ of forskolin and 20 μΜ of genistein were added to the cells. Under our recording conditions, the current density after hours of incubation at 27 ° C was higher than that observed after 24 hours of incubation at 37 ° C. These results are compatible with the known effects of low temperature incubation on the density of AF508-CFTR in the piasmatic membrane. To determine the effects of

<img file="AR080944A1_D0267.tif" />

149 correction compounds on the current density d

CFTR, the cells were incubated with 10 μΜ of the test compound for 24 hours at 37 ° C and the current density was compared with the controls of 27 ° C and 37 ° C (% activity). Prior to registration, the cells were washed 3X with extracellular recording medium to remove any remaining test compound. Preincubation with 10 μΜ of correction compounds significantly increased the cAMP and genistein-dependent current compared to the controls at 37 ° C.

Identification of enhancer compounds [00315] The capacity of ÄF508-CFTR enhancers to increase macroscopic current AF508-CFTR Cl '(I) was also investigated_fiF508) in NIH3T3 cells stably expressing ÛF508-CFTR by perforated patch registration techniques. The enhancers identified from the optical tests caused by a dose-dependent increase in Ιλι ·· 508 with similar potency and efficiency to that observed in the optical tests. In all the cells examined, the investment potential before and during the application of the enhancer was around -30 mV, which is the calculated ECi (-28 mV).

Solutions [00316] Intracellular solution (in mM): Cs aspartate (90), CsCl (50), MgCl₂ (1), HEPES (10), and 240 pg / ml amphotericin-B (pH adjusted to 7.35 with CsOH).

150

<img file="AR080944A1_D0268.tif" />

(00317] Extracellular solution (NMDG) -Cl (150), MgCl₂ (2), adjusted to 7.35 with HCl).

Cell culture

<img file="AR080944A1_D0269.tif" />

[00318] NIH3T3 mouse fibroblasts that are stably expressing AF508-CFTR are used for whole cell records. The cells are kept at 37^?C in 5% CO₂ and

90% moisture in Dulbecco's modified Eagle medium supplemented with 2 mM glutamine, 10% fetal bovine serum, 1 X NEAA, ß-ME, 1 X pen / estrep and 25 mM HEPES in 175 cm culture flasks². In all the complete cell registers, 2,500-5,000 cells were seeded in glass coverslips coated with poly-L-lysine and cultured for 24-48 hours at 27 ° C before using to test the activity of the enhancers; and incubated with or without the correction compound at 37 ° C to measure the activity of the correctors.

Single Channel Records [00319] The unique channel activities of

Temperature-corrected AF508-CFTR expressed as stable in NIH3T3 cells and the activities of enhancer compounds using membrane patch cleaved from the inside out. In short, the voltage clamp records of the single channel activity were performed at room temperature with an Axopatch 200B zonal clamp amplifier (Axon Instruments Ine.).

<img file="AR080944A1_D0270.tif" />

151

All records were obtained with a sampling frequency of 10 kHz and a low-pass filter at 400 Hz. The pipettes were made of Corning Kovar Sealing # 7052 glass (World Précision Instruments, Inc., Sarasota, FL) and had a resistance of 5-8 Ω when loaded with the intracellular solution. AF508-CFTR was activated after cleavage, by adding 1 mM Mg-ATP and 75 nM of the catalytic subunit of the protein kinase-dependent kinase

CAMP, (PKA; Promega Corp. Madison, Wl). After stabilizing the channel activity, the patch was perfused using a gravity-operated perfusion system. The influx was placed adjacent to the patch, resulting in the complete exchange of the solution within 1-2 seconds. To maintain ZTR activity of CFTR during rapid perfusion, the non-specific phosphatase inhibitor F "(10 mM NaF) was added to the barium solution. Under these registration conditions, the channel activity remained constant throughout the duration of the patch patch (up to 60 minutes). The currents produced by the positive charges that move from intracellular to extracellular solutions (anions that move in the opposite direction) are shown as positive currents. The pipette potential (V_p) was maintained at 80 mV.

100320] Channel activity of membrane patches containing &lt;2 active channels was analyzed. The maximum number of simultaneous holes determined the number of channels

152

<img file="AR080944A1_D0271.tif" />

active during the experiment. To determine the amplitwd of single channel current, the 120-second data recorded from the CFTR AF508 activity was filtered off-line at 100 Hz and then used to construct total point amplitude histograms that were adjusted with multigaussian functions using the Bio-Patch Analysis (Bio-Logic Comp., France) computer program. Total microscopic current and open probability (P_or) were determined from 120 seconds of channel activity. The P_or was determined using the Bio-Patch computer program or the P ratio_or = I / i (N), where I = average current, i = single channel current amplitude and N = number of active channels in the patch.

Solutions [00321] Extracellular solution (in mM): NMDG (150), aspiric acid (150), CaCl₂ (5), MgCl₂ (2), and HEPES (10) (pH adjusted to 7.35 with Tris base).

[00322] Intracellular solution (in mM): NMDG-Cl (150),

MgCl₂ (2), EGTA (5), TES (10), and Tris base (14) (pH adjusted to 7.35 with HCI).

Cell culture [00323] Fibroblasts from NIH3T3 mice stably expressing AF508-CFTR are used for membrane cleaved zonal clamp registers. The cells are kept at 37^andC in 5% CO₂ and 90% humidity in medium

Modified Dulbecco Eagle supplemented with 2 mM of

<img file="AR080944A1_D0272.tif" />

153 glutamine, 10% fetal bovine serum, 1 X NEAA, β-ME, 1 X pen / estrep, and 25 mM HEPES in 175 cm culture flasks². In the single channel records, 2,5005,000 cells were seeded in glass coverslips coated with poly-L-lysine and cultured for 24-48 hours at 27 ° C before use.

[00324] Using the procedures described above, activity (EC) was measured.₅o) of compound 2 and is shown in the

Table 2-2

Table 2-2

IC50 / EC50 ree .: + + + &lt;- 2,0 &lt;+ + &lt;= 5,0 &lt;+% of ree activity: + &lt;= 2 5,0 &lt;

++ &lt;- 100,0 &lt; +++

Max

Comp.

EC50 ree. ree efficiency

compound [00325] Using the procedures described above, activity (EC) was measured₅₀) of compound 3 and is shown in the

Table 3-2

Table 3-2

IC50 / EC50 ree .: +++ &lt;= 2,0 &lt;++ &lt;=

<img file="AR080944A1_D0273.tif" />

154 % of activity ree + &lt;- 25.0 &lt;

5,0 &lt; +

<img file="AR080944A1_D0274.tif" />

++ &lt;= 100,0 &lt; +++

Comp.

EC50 ree

Max compound efficacy

OTHER FORMS OF EMBODIMENT [00326] All publications and patents mentioned in this description are hereby incorporated by reference to the same degree as if each individual patent publication or application had been specifically and individually indicated as incorporated by reference. If the meaning of the terms of any of the patents or publications incorporated by reference conflicts with the meaning of the terms used in this description, the meaning of the terms in this description is dominant.

On the other hand, the above treatment merely reveals and describes exemplary embodiments of the present invention. A person skilled in the art will readily recognize from such treatment and the accompanying drawings and claims that various, changes, modifications and variations can be made herein without departing from the spirit and scope of the invention such as defined in the following claims.