TW201204720A - Pharmaceutical compositions and administrations thereof - Google Patents

Abstract

The present invention relates to pharmaceutical compositions comprising a compound of Formula I in combination with one or both of a Compound of Formula II and/or a Compound of Formula III. The invention also relates to pharmaceutical formulations thereof, and to methods of using such compositions in the treatment of CFTR mediated diseases, particularly cystic fibrosis.

Description

201204720 VI. OBJECTS OF THE INVENTION: TECHNICAL FIELD The present invention relates to a pharmaceutical composition comprising one or both of a compound of formula I and a compound of formula π and/or a compound of formula III. The invention is also directed to pharmaceutical formulations thereof and methods of using the compositions to treat CFTR-mediated diseases, particularly cystic fibrosis. The present application claims priority to U.S. Provisional Application Serial No. 61/327,095, filed on Apr. 22, 2010. The entire content of this priority application is incorporated herein by reference. [Prior Art] Cystic fibrosis (CF) is a recessive hereditary disease that affects approximately 30,000 children and adults in the United States and affects approximately 30,000 children and adults in Europe. Despite advances in the treatment of CF, the disease cannot be cured. The CF line is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that encodes a gene responsible for the regulation of salt and water uptake and secretion of epithelial chloride channels in various tissues. A small molecule drug called a potentiator that increases the probability of CFTR channel opening represents a potential therapeutic strategy for treating CF. A synergist of this type is disclosed in WO 2006/002421, which is incorporated herein in its entirety by reference. Another potential therapeutic strategy involves small molecule drugs called CF correctors that increase the number and function of CFTR channels. A corrective agent of this type is disclosed in WO 2005/075435, which is incorporated herein in its entirety by reference. In particular, CFTR is a cAMP/ATP-mediated anion channel that is expressed in a variety of cell types, including absorption and secretion of 155839.doc 201204720 dermal cells, which regulates anionic transmembrane flow and other ion channel and protein activity^ The normal function of CFTR in epithelial cells is critical for maintaining systemic (including respiratory and digestive tissue) electrolyte transport. CFTR is composed of approximately 1480 amino acids encoding a protein consisting of adaptor repeats of the transmembrane domain, each transmembrane domain containing six transmembrane helix and nucleotide binding domains. The two transmembrane domains are joined by a large polar regulatory (R)-domain with multiple phosphorylation sites that regulate channel activity and cellular trafficking. The gene encoding CFTR has been identified and sequenced (see Greg〇ry, R. et al., (1990) Nature 347: 382-386; Rich, D. Ρ et al., (1990) Nature 347: 358-362), (Riordan, JR et al., (1989) Science

245: 1066-1073). Defects in this gene cause CFTR mutations, which cause cystic fibrosis ("CF")' cystic fibrosis to be the most common fatal human genetic disease. In the United States, about 1 in every 2, 5 babies have cystic fibrosis. In the general American population, up to 10,000 people carry a single copy of the defective gene without significant disease effects. In contrast, individuals with two copies of the CF-related gene suffer from the debilitating and lethal effects of CF, including chronic lung disease. In patients with CF, CFTR mutations that are endogenously expressed in the airway epithelium cause a decrease in pericardial anion secretion, resulting in an imbalance of ion and fluid transport. The reduction in anion transport caused by the increase in mucus accumulation in the lungs and the accompanying microbial infection eventually resulted in the death of $CF patients. In addition to respiratory diseases, CF patients often have gastrointestinal problems and pancreatic function. If not treated, it will lead to death. In addition, most men with cystic fibrosis are infertile and fertility is reduced in women with cystic fibrosis. Contrary to the severe effects of two copies of the CF-related gene, individuals with a single copy of the CF-related gene showed increased resistance to cholera and dehydration caused by diarrhea - perhaps indicating a relatively high incidence of CF genes in the population . Sequence analysis of the CFTR gene of the CF chromosome reveals a variety of diseases that cause mutations (Cutting, GR #A' (1990) Nature 346: 366-369; Dean, Μ. et al., (1990) Cell 61: 863: 870; And Kerem, B-S_ et al., (1989) Science 245:1073-1080; Kerem, BS et al., (1990) Proc. Natl. Acad. Sci. USA 87:8447-8451). To date, more than 1,000 diseases that cause mutations in the CF gene have been identified (http://www.genet.sickkids.on.ca/cftr7app). The most common mutation is the loss of phenylalanine at position 508 of the CFTR amino acid sequence and is often referred to as ΔF5 08-CFTR. This mutation occurs in approximately 70% of cases of cystic fibrosis and can lead to serious disease. Deletion of residue 508 in ΔF508-CFTR inhibits accurate folding of the nascent protein. This leaves the mutant protein unable to leave the ER and migrate to the plasma membrane. Thus, the number of channels present in the membrane is much less than that observed in cells expressing wild-type CFTR. In addition to the impaired migration, mutations also cause defective channel gating. In summary, reduced number of channels in the membrane and defective gate control result in reduced trans-epithelial transport of anions, resulting in defective ion and fluid transport (Quinton, P. M. (1990), FASEB J. 4: 2709-2727). However, studies have shown that the number of reduced AFSOS-CFTR in the membrane is still functional, 155839.doc 201204720 but less functional than wild-type CFTR (Dalemans et al, (1991), Nature Lond. 354: 526-528; Denning et al, above) Pasyk and Foskett (1995), J. Cell. Biochem. 270: 12347-50). In addition to AFSOS-CFTR, other diseases that cause CFTR mutations to initiate defective migration, synthesis, and/or channel gating can be up- or down-regulated to alter anion secretion and improve disease progression and/or severity. Although CFTR transports a variety of molecules in addition to anions, it is apparent that this effect (transport anion) indicates that it is one of the important mechanisms for transporting ions and water across the epithelium. Other components include the epithelial Na+ channel, the ENaC, Na+/2C1_/K+ cotransporter, the Na+-K+-ATPase pump, and the basal lateral membrane K+ channel, which are responsible for the absorption of gas ions into the cells. These elements work together to achieve trans-epithelial directed transport via their selective expression and localization within the cell. Gas ion absorption was carried out under the coordinated activities of ENaC and CFTR present on the apical membrane and the Na+-K+-ATPase pump and Cl_ ion channel on the outer surface of the cell basal. Secondary active transport of chloride ions from the cell lumen side causes intracellular gas ion accumulation, which can then passively exit the cell via the Cl_ channel, causing vector transport. The placement of the Na+/2C1_/K+ cotransporter, the Na+-K+-ATPase pump and the basolateral membrane K+ channel on the basolateral surface and the CFTR coordinated gas ion on the cell lumen side were secreted via CFTR on the cell lumen side. Since water itself may never be actively transported, it relies on a small transepithelial osmotic gradient generated by the overall flow of sodium and chloride ions across the epithelial flow. As discussed above, the deletion of residue 508 in Xianxin AF508-CFTR inhibits the accurate folding of the nascent protein such that the mutant protein cannot leave the ER and migrate 155839.doc 201204720 to the plasma membrane. Therefore, an insufficient amount of mature protein is present at the plasma membrane and gas ion transport in the epithelial tissue is significantly reduced. In fact, this cellular phenomenon of defective ER treatment of ABC transporters by ER institutions has demonstrated not only the basic principles of CF disease, but also the basic principles of a wide range of other isolated and hereditary diseases. Compounds that are potentiators of CFTR proteins (such as compounds of formula I) and compounds that are corrective agents for CFTR proteins, such as compounds of formula II or formula III, have independently been shown to have a therapeutic disorder of CFTR (such as cystic fibrosis). The effect. Thus, there is a need for novel treatments for CFTR-mediated diseases involving CFTR correctors and potentiator compounds. In particular, there is a need for combination therapies for the treatment of CFTR mediated diseases, such as cystic fibrosis, which include CFTR potentiators and corrective compounds. More particularly, there is a need for combination therapies for the treatment of CFTR-mediated diseases, such as cystic fibrosis, comprising a CFTR potentiator compound (such as a compound of Formula I) and a CFTR Corrector compound (such as Formula II and/or Formula III) Compound). More specifically, there is a need for combination therapies for the treatment of CFTR-mediated diseases, such as cystic fibrosis, comprising a CFTR potentiator compound (such as Compound 1) and a CFTR corrective compound (such as Compound 2 and/or Compound 3) . SUMMARY OF THE INVENTION To meet these and other needs by the present invention, the present invention is directed to a pharmaceutical composition comprising: a compound of formula I 155839.doc 201204720

Or a pharmaceutically acceptable salt thereof, wherein: Ring A is selected from the group consisting of:

(b)

X

or

(c) (d) R1 is -CF3, -CN or -C tri CCH2N(CH3)2; R2 is hydrogen, -CH3, -CF3, -OH or -CH2OH; R3 is hydrogen, -CH3, -OCH3 or - CN ; The restriction condition is that R2 and R3 are not nitrogen at the same time; and one or both of the following:

A compound of the formula II or a pharmaceutically acceptable salt thereof, wherein: T is -CH2-, -CH2CH2-, -CF2-, -c(ch3)2- or -c(o)-; R!' is Η, Cu aliphatic group, halogen group, CF3, CHF2, 0 ((:, -6 month family 155839.doc -9- 201204720 base); and RD1 or R02 is ZDR9, wherein: ZD is a bond, CONH, S02NH, SC^l^Cw alkyl), CH2NHS02, CH2N(CH3)S02, CH2NHCO, COO, S02 or CO; and R9 is H, Cw aliphatic or aryl; and/or a compound of formula III

Or a pharmaceutically acceptable salt thereof, wherein: R is hydrazine, OH, OCH3, or two R forms together - 〇CH20- or -OCF20-, R4 is hydrazine or alkyl; R5 is hydrazine or F; 11 or CN; R7 is Η, -CH2CH(OH)CH2OH, -CH2CH2N+(CH3)3 or -CH2CH2OH; 118 is 11, OH, -CH2CH(OH)CH2OH, -ch2oh, or r7 and r8 together form a five-membered ring . In another aspect, the pharmaceutical composition comprises Compound 1, 155839.doc • 10-201204720

Compound 1 and Compound 2 and/or Compound 3.

Compound 3 In one aspect the pharmaceutical composition comprises Compound 1, Compound 2 and Compound 3. In another aspect, the invention is directed to a pharmaceutical composition comprising at least one component from column A of Table I and at least one component from column 3 and/or c of Table 1. These components are described as corresponding embodiments of the invention in the corresponding paragraphs of the following pages. For convenience, Table I describes the paragraph numbers and corresponding headings of the examples of the compounds. For example, the embodiment of the hydrazine compound is disclosed in paragraph Α.Α.1 of the present specification. 155839.doc -11 - 201204720 Table i JA block f Example JB block exemption 1 C block Example paragraph heading paragraph heading paragraph heading II.A.1. Formula I compound II.B.1 · Formula II compound II .C.1. Compound of Formula III II.A.2. Compound 1 II.B.2. Compound 2 II.C.2. Compound 3 In one aspect, the present invention comprises a pharmaceutical composition comprising a selected from the group consisting of The components of any of the examples described in Table A, and the components of any of the embodiments described in column B of Table I and/or any of the embodiments selected from column C of Table I The components. Thus, in one embodiment, the invention is directed to a pharmaceutical composition comprising a compound of formula I and a compound of formula II. In another embodiment, the present invention is directed to a pharmaceutical composition comprising a compound of formula I and a compound of formula III. In another embodiment, the present invention is directed to a pharmaceutical composition comprising a compound of formula I, a compound of formula II, and a compound of formula III. In another embodiment, the present invention is directed to a pharmaceutical composition comprising Compound 1 and a compound of Formula II. In another embodiment, the present invention is directed to a pharmaceutical composition comprising Compound 1 and a compound of Formula III. In another embodiment, the present invention is directed to a pharmaceutical composition comprising Compound 1, a compound of Formula II, and a compound of Formula III. In another embodiment, the present invention is directed to a pharmaceutical composition comprising Compound 1 and Compound 2. In another embodiment, the present invention is directed to a pharmaceutical composition comprising Compound 1 and Compound 3 in a package 155839.doc -12-201204720. In another embodiment, the present invention is directed to a pharmaceutical composition comprising Compound 1, Compound 2, and Compound 3. In another embodiment, the present invention is directed to a pharmaceutical composition comprising a compound of formula I and compound 2. In another embodiment, the present invention is directed to a pharmaceutical composition comprising a compound of formula I and compound 3. In another embodiment the invention relates to a pharmaceutical composition comprising a compound of formula I, compound 2 and compound 3. The various components listed in Table I have been disclosed and can be found in U.S. Patent No. 7,776,905, U.S. Patent No. 7,645,789, U.S. Patent Application Serial No. 2010/01, No. The contents of these patents are incorporated herein by reference in their entirety by reference. [Embodiment] I. Definitions Unless otherwise indicated, the following definitions as used herein shall apply. The term "ABC-transporter" as used herein means an ABC-transporter or a fragment thereof comprising at least one binding domain, wherein the protein or fragment thereof is present in vivo or ex vivo. The term "binding domain j" as used herein means a domain which can bind to a modulator on an ABC-transporter. See, for example, Hwang, T. C. et al., J. Gen. Physiol. (1998): 7" 3), 477-90 ° 155839.doc -13 - 201204720 The term "CFTR" as used herein means a cystic fibrosis transmembrane conductance regulator or a mutant thereof capable of exerting regulatory factor activity, including but not limited to ^508 CFTR, R117H CFTR and G551D CFTR (for CFTR mutants see, for example, http://www.genet.sickkids.on.ca/cftr/). The term "active pharmaceutical ingredient" or "r API" as used herein refers to a biologically active compound. Exemplary APIs include the CF synergist N-(4-(7-azabicyclo[2.2.1]hept-7-yl)-2-(trifluoromethyl)phenyl)-4- oxo-5 -(Trifluoromethyl)-1,4-indoline-3 -cartoamine (Compound 1). Exemplary APIs also include the CF correcting agent 3-(6-(1-(2,2-difluorobenzo[d][l,3]dioxol-5-yl)cyclopropanoguanidino) -3-methylpyridin-2-yl)benzoic acid (compound 2) and (/〇-1·(2,2-difluoro benzo[d][l,3]dioxol-5- -N-(1-(2,3-dihydroxypropyl)-6-fluoro-2-(1-hydroxy-2-mercaptopropan-2-yl)-lH-indole η多·5_ base Cyclopropanol (Compound 3). The term "modulate" as used herein means to increase or decrease the amount that can be measured. As used herein, the term "normal CFTR" or "normal CFTR function" means no A wild-type CFTR that is damaged by environmental factors such as smoking, contamination, or anything that causes inflammation in the lungs. As used herein, the term "reduced CFTR" or "reduced CFTr# can mean less than Normal CFTR or less than normal CFTR function. As used herein, the term "amorphous" refers to a solid material that has no long range order in terms of molecular position. Amorphous solids are generally ultra-cold liquids where the molecules are random Arranged so that there is no limit 155839.doc -14· 2012 04720 distinct arrangements (eg molecular packing) ^ no long-range order. Amorphous solids are generally isotropic, ie exhibit similar properties in all directions and have no solid melting point. For example, amorphous materials are In the X-ray powder diffraction (X-June) diagram, there is no sharp characteristic crystallization peak (that is, the crystal is not crystallized as determined by Yan Qing). In fact, 'one or several appear in its xrpd diagram. Broad peaks (eg, South base). Broad peaks are characteristic of amorphous solids. For a comparison of xrpd of amorphous and crystalline materials, see still 2004/0006237. The term "substantially amorphous" is used herein. Refers to a solid material that has almost no long-range order in terms of molecular position. For example, a substantially amorphous material has a crystallinity of about 15% or less (for example, about (10) or less: crystallinity or about 5% or less. Itching, ._ ^ ^, 'mouth day and day.' It should also be noted that the term "substantially amorphous" includes the descriptive word "non-曰 you. Non-Japanese shape", the latter means no (0%) crystallinity The term "dispersion" as used herein means A substance (ie, a dispersed phase) is a dispersion system that is distributed in individual units throughout the second substance f (continuous phase or vehicle). The size of the dispersed phase can be quite different (eg, single-molecule, nano-sized colloidal particles) In many cases, the two phases can be solid, liquid or gas. In the case of a solid dispersion, both the dispersed phase and the continuous phase are solid. In the application of the drug, the solid dispersion A solid dispersion of amorphous music in an amorphous polymer; a solid dispersion of an amorphous material in a crystalline polymer; a crystalline drug in an amorphous form. a solid dispersion in the product; or a solid bear dispersion of the crystalline drug in the crystalline polymer. In the present invention, the solid dispersion may comprise a solid dispersion of the amorphous drug at 155839.doc 201204720, which is a solid dispersion of solid or amorphous bismuth. A polymer in which the material is dispersed in the polymer, and the N:: solid dispersion comprises a composition comprising a nucleus comprising a dispersed phase = a continuous phase. Alternatively, the solid dispersion package is a piece of music and the polymer constitutes a continuous phase. Word; "Dispersion" means two or two (four) and polymer (4) containing: two or other pharmaceutical excipients) and two (such as a surfactant) are substantially amorphous (eg, = music) (eg, compound /, having a force of 1 5% or less (for example, about 10% or less, or about 5% or less) of crystalline drug (9) such as N_(4-(7-aza) Bicyclo [2 2 11* 7 甘, 1 .]heptyl _ yl)-2_(trifluoromethyl)phenyl)-4- oxonite (-fluoromethyl)-1,4_dichloroindene _ 3_N-branched amine)) or amorphous (also 'ruthenium drug') and the physical properties of the substantially amorphous or amorphous drug are enhanced and/or / and the solubility is enhanced by other components. L is a solid dispersion in which a compound is dispersed in a suitable carrier medium such as a solid carrier. For example, the carrier comprises a polymer (eg, a water soluble polymer or a partially water soluble polymer), and Excipients may optionally be included, such as a dysfunctional excipient (eg, or a plurality of surfactants) or a non-functional fugitive J (eg, 'one or more fillers') q-exemplary solid dispersion The body is N-(4-(7- Heterobicyclo[2.2.1]hept-7-yl)-2-(trifluoromethyl)phenyl)-4-yloxy-5-(trifluoromethyl)_ls4•dihydroquinoline_3_曱a coprecipitate or co- spheroid of at least one polymer. A "co-precipitate" is a product obtained by dissolving a drug and a polymer in a solvent or a solvent mixture, followed by removal of the solvent or solvent mixture. 155839.doc -16- 201204720 The polymer is suspended in a solvent or solvent mixture. The solvent or solvent mixture includes organic solvents and supercritical fluids. "Compresses" are drugs and polymerizations in the presence of a solvent or solvent mixture, as appropriate. Heating to melt, followed by mixing 'products that remove at least a portion of the solvent when appropriate and are cooled to room temperature at a selected rate. As used herein, "crystalline" means that the structural units are arranged in a fixed geometric pattern or ruthenium to crystallize solids. A compound or composition having a strictly long-range order. The structural unit constituting the crystal structure may be an atom, a molecule or an ion. The crystalline solid exhibits a fixed melting point. As used herein, the phrase "substantially crystallized" means pattern Or a solid matter arranged in a lattice and having a strict long-range order. For example, a substantially crystalline material has a crystallinity of about 85% or more (for example, a crystallinity of about 90% or more or about 9% or more). At the end of the day, please also note that the term "substantially includes, & Η ', ° day", the latter is defined in the previous paragraph. "Crystallinity" refers to the structural order of a solid. For example, a 5 'substantially amorphous compound 丨 has a crystallinity of about 15% or less' or a solid structure of about 15% or less. In another example, Amorphous Compound 1 has zero (0%) crystallinity. Examples of the inactive excipients used in the pharmaceutical compositions include fillers or diluents, binding agents, slip agents, lubricants, disintegrating agents, and the like. As used herein, a "disintegrant" tablet dispersible excipient. Disintegrant Examples of hydrating and contributing to the pharmaceutical composition include croscarmellose sodium 155839.doc • 17· 201204720 and/or sodium starch glycolate. As used herein, "diluent diluent" or filler" is used to increase the bulkiness of a pharmaceutical composition. Examples of fillers include

Sugar, sorbitol, cellulose, calcined gA, starch, sugar (e.g., mannitol, sucrose, or the like), or any combination thereof. As used herein, "surfactant - W" is an excipient that imparts enhanced solubility and/or wettability to a pharmaceutical composition. Examples of surfactants include sodium sulfonate (SLS), sodium succinate, sodium succinate (ssf), polyoxyethylene 20 sorbitan monooleate (eg, TweenTM), or any combination thereof. 0. A mildew agent as used herein is an excipient that imparts enhanced cohesion or tensile strength (e.g., hardness) to a pharmaceutical composition. Examples of the binder include bismuth sulphate, sucrose, corn starch, microcrystalline cellulose, and modified cellulose (e.g., hydroxymethylcellulose). A "sliding agent" as used herein is an excipient that imparts enhanced flow characteristics to a pharmaceutical composition. Examples of the slip agent include colloidal cerium oxide and/or talc. As used herein, "colorant" is an excipient that imparts the desired color to a pharmaceutical composition. Examples of the coloring agent include commercially available pigments such as ?13&(:Blue No. 1 Aluminum Lake, FD&C Blue No. 2, other FD&c blue, titanium oxide, iron oxide and/or Or a combination thereof. "Lubricant" as used herein is an excipient that is added to a pharmaceutical composition that will be compressed into a tablet. The lubricant helps to compress the granules into tablets and the tablets of the pharmaceutical composition. Demoulding in a die press. Example of Lubricant 155839.doc 201204720 Includes stearyl magnesium, stearate, or any combination thereof. 曰), oxidized oil, stearin anti-butyl Guarantee = = :::= means that despite the application of external pressure, the tablet still has the mathematical expression = characteristic 1 can use coffee

% Brittleness = 100 X K (1) : Medium:: is the original weight of the spinning agent' and % is the final weight of the tablet after passing through the fraguator. ^ The standard coffee test device measures the brittleness, and the test device flips the experiment. Some of the lozenges of the present invention have a brittleness of about (e.g., about 0.75 /. or less, about 5% or less or less than about 〇). As used herein, "average particle diameter" is the average particle diameter as measured using techniques such as laser light scattering, image analysis or sieving analysis. As used herein, "grain density" is the mass of a particle of a substance divided by the total volume occupied by the particle. The total volume includes particle volume, interparticle volume, and internal pore volume. Bulk density is not an inherent property of matter f; it varies depending on how the material is handled. The term "aliphatic" or "aliphatic" as used herein, means a straight chain (ie, no branching bond) or a branched chain, substituted or unsubstituted, which is fully saturated or contains - or more unsaturated units. a hydrocarbon chain' or a monocyclic or bicyclic hydrocarbon that is fully saturated or contains one or more units of unsaturation, but is not aromatic (also referred to herein as "carbon ring", "cycloaliphatic" or "cycloalkyl" ), which has a single point of attachment to the rest of the molecule. Unless otherwise stated, the aliphatic group contains 20 aliphatic carbon atoms. In some embodiments, the aliphatic group contains a group of 155839.doc 19 201204720 carbon atoms. In other embodiments, the aliphatic group contains from 1 to 8 aliphatic carbon atoms. In other embodiments, the aliphatic group contains from 1 to 6 aliphatic carbon atoms, and in other embodiments the 'aliphatic group contains from 1 to 4 aliphatic carbon atoms. In one embodiment, 'cycloaliphatic" (or "carbocyclic" or "cycloalkyl") refers to a monocyclic C3_C8 hydrocarbon that is fully saturated or contains one or more units of unsaturation but is not aromatic. Or a bicyclic or tricyclic cvcm hydrocarbon having a single point of attachment to the remainder of the molecule, wherein any individual ring in the bicyclic system has 3-7 members. Suitable aliphatic groups include, but are not limited to, straight or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl, and mixtures thereof, such as (cycloalkyl)alkyl, (cycloalkenyl) Alkyl or (cycloalkyl)alkenyl. Suitable cycloaliphatic groups include cycloalkyl, bicyclic cycloalkyl (eg decahydronaphthalene), bridged bicycloalkyl (such as norbornyl or [2.2. 2]bicyclooctyl), or bridged tricyclic ( Such as gold ^ _ used herein § my "heteroaliphatic" means one or two carbon atoms independent of polyoxo, m-cut replacement of aliphatic groups. Heteroaliphatic == or unsubstituted, branched or none Branching, cyclic or "heterocyclic", heterocyclic, "heterocyclic" or "heterocyclic" as used herein means "more than 1 g heterocycloaliphatic" or heterocyclic a ring member, wherein one = aliphatic or "heterocyclic" group has 3 to 杂 or a hetero atom of the lining, and the " bicyclic member is independently selected from the ring of the oxygen, sulfur, and nitrogen systems. 3 to 7 ring members. Aromatic, monocyclic,: members are non-,, ":1 bicyclic or tricyclic systems of independently selected heteroatoms. In some embodiments, "Miscellaneous 155839.doc 201204720 Terminology "Hetero atom" means one or more of oxygen, sulfur, a, lin or Shi Xi (including nitrogen, sulfur, antimony or Shi Xi, any of the emulsified open / formula, any of the four types of nitrogen a substituted form; or a heterocyclic ring which may be substituted with a nitrogen such as N (as in 3,4-diaza 2//piroxime) NH (as in Bihadyl) or as + (as in n) The upper substituted thiophene is used in the base;)). As used herein, the term "unsaturated" means having - or a portion of a plurality of unsaturated units. Separately or as a larger part (eg, in "aroze" The term "aryl" as used in the "base", "aryl oxy" or "square aryl" group, is used to refer to the early piers and double rings of the star with a total of 5 to 14 ring members. A tricyclic system in which at least one ring in the system is an aromatic ring and wherein each ring in the system contains (1) ring members. The term "aryl" is used interchangeably with the term "aryl ring". The term "square" Also referred to as a heteroaryl ring system as defined below. An aliphatic or heteroaliphatic group or a non-aromatic heterocyclic ring may contain one or more substituents. Aliphatic or heteroaliphatic groups or non-aromatic heterocycles Suitable substituents on the saturated carbon are selected from the above substituents for the unsaturated carbon (IV) of the aryl or heteroaryl group and additionally include the following: = 0, = s, hr, = nn (r.) 2, =丽 HC(9)R., "Lan HC〇2 (alkyl), = 丽基基) or NR wherein each R is independently selected from hydrogen or optionally substituted a 6 aliphatic. Aliphatic R. The substituents which are optionally present are selected from the group consisting of nh2, NH(Cl·^ group), N(Cl.4 aliphatic group) 2, self group, aliphatic group, OH, hydrazine (Cl. 4 aliphatic group), N〇2, CN, CO#, c〇U is a group of a group), 〇 (donyl Cl_4 aliphatic group) or a dentate group (c] 4 aliphatic group), wherein each of the above (3 ** 4 aliphatic groups) R· is unsubstituted. 155839.doc -21 - 201204720

The substituents present on the gas of the non-aromatic heterocyclic ring are selected from _r+, -N(R+)2^-C(0)R+ . .C02R+ . .C(〇)C(〇)R+ ^ _C (0)CH2C _+, -S〇2R+, ·δ〇2Ν(ΙΙ+)2, _c(=s)N(R+)2, _C(=NH)·N(R+)2 or pendulum+s〇2R+ Wherein R + is hydrogen, optionally substituted by a Cl 6 aliphatic group, optionally substituted phenyl, optionally substituted _o (ph), optionally substituted -CH 2 (Ph), optionally Substituted _(CH2)1 2(ph); optionally substituted -CH=CH(Ph); or unsubstituted 5 having 丨 to 4 heteroatoms independently selected from oxygen, nitrogen or sulfur To 6 members heteroaryl or heterocyclic, or although in accordance with the above definition, two independently present R+ on the same substituent or different substituents together with the atoms bonded to each r+ group form 0 to 3 independent A 3- to 8-membered cyclod, heterocyclic, aryl or heteroaryl ring selected from the group consisting of 4, oxygen or sulfur heteroatoms. The substituents which are optionally present on the aliphatic or phenyl ring R+ are selected from the group consisting of NH2, Nh (Ci-4 aliphatic), the group of the group 2, a functional group, a Cl 4 aliphatic group, 〇H, 〇 ( Ci4 aliphatic group), N〇2, CN C02H, C02 (CN4 aliphatic group), anthracene (functional Ci 4 aliphatic group) or a dentate group (C!·4 aliphatic group), wherein each of the aforementioned aliphatic groups The base r+ is unsubstituted. As detailed above, in some embodiments, two independently present R' (or similar herein; any other variable of t), together with the atoms to which the variables are combined, form from 10 to 3 independently selected from nitrogen. a 3- to 8-membered cycloalkyl, heterocyclyl, aryl or heteroaryl ring of a hetero atom of oxygen or sulfur. An exemplary ring formed when two independently existing Rs (or any other variables similarly defined herein), together with the atoms of each variable, include (but are not limited to) the following. a) Two independent existences R, (or any other variable similarly defined herein) binds to the same atom and forms a ring along with the other atom, such as N(R,)2, which has two R's along with a nitrogen atom in 155839.doc •22·201204720 Forming a maiden _ 丨 基 基 基 基 或 or morpholin-4-yl; and b) two independently existing stalks, (or any other variable similarly defined herein) bound to a different atom and together with 0 is formed into a ring, for example, in the presence of two enthalpies of phenyl through R, substituting γ, two present ^>0 together with the oxygen atom to which they are combined to form a fused 6-membered, OR〇-oxygen ring: A plurality of other rings may be formed when two independently present R, (or any other variable similarly defined herein), together with the atoms to which the various variables are combined, and the examples detailed above are not intended to be limiting. A substituent, as exemplified below, bonded to, for example, a bicyclic system, means that the substituent can be attached to any substitutable ring atom on either ring of the bicyclic ring system:

The term "protecting group" (PG) as used herein denotes a group intended to protect a functional group (such as an alcohol, an amine, a buffer, a few groups, etc.) against an undesirable reaction during the synthetic procedure. Commonly used protecting groups are disclosed in Greene and Wuts, Protective Groups in Organic Synthesis > (John

Wlley & Sons, New York, 1999) is incorporated herein by reference. Examples of the nitrogen protecting group include an anthracenyl group, an arylsulfonyl group or an amine carbenyl group such as a decyl group, an ethyl fluorenyl group, a propyl fluorenyl group, a pentylene group, a tert-butyl butyl group, and a 2-chloroethyl group. , 2_bromoethenyl, trifluoroethylidene, trichloroethane, ugly, o-nitrophenyloxyethyl, acetobutyl, benzoquinone, 4-chlorophenyl fluorenyl , 4-bromobenzoyl, 4-nitrobenzylidene and a palmitic auxiliary such as protected or unprotected D-amino acid, L-amino 155839.doc -23- 201204720 acid Or D, L-amino acid 'such as alanine, leucine, phenylalanine and the like; sulfonyl, such as phenylsulfonyl, p-toluenesulfonyl and the like; urethane Ester group, such as benzyloxycarbonyl, p-benzobenzyloxy, p-nonyl phenyl hydroxycarbonyl, p-nitrobenzyloxycarbonyl, 2-nitrophenyl hydroxycarbonyl, p-bromobenzene Oxyloxycarbonyl, 3,4-dioxaoxybenzethoxycarbonyl, 3,5-dimethoxybenzoyloxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl, 4-methyl Oxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzoquinoneoxy , 3,4,5-trimethoxybenzophenoxycarbonyl, 丨-(p-biphenyl)-1-methylethoxycarbonyl, α,α-dimethyl-3,5-dimethoxybenzene Methoxy, diphenylphosphonium oxycarbonyl, tert-butoxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl, 2,2,2·trisethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxycarbonyl, -9-fluorenyloxycarbonyl, cyclopentyloxycarbonyl, adamantyloxycarbonyl, ring a hexyloxycarbonyl group, a phenylthiocarbonyl group and the like, an arylalkyl group such as a benzyl group, a triphenyl fluorenyl group, a benzyloxy fluorenyl group and the like, and a terracotta group, such as three Methyl fluorenyl and the like. Preferably, the protecting group is a third butoxycarbonyl group (B〇c). Examples of suitable protecting groups for the acid are substituted alkyl esters such as 9-fluorenylmethyl, methoxymethyl, methylthiomethyl, tetrahydro-decyl, tetrahydrofurfuryl, a Oxyethoxymethyl, 2-(trimethyldecyl)ethoxymethyl, benzyloxymethyl, pentyloxymethyl, phenylethenyloxymethyl, triisopropyl Basestone, methyl, cyanomethyl, propyl-alcohol, benzoyl thiol, substituted benzamidine methyl ester, 2,2,2·tris, ethyl, 2-dentylethyl, alkane Base, 2-(trimethyldecyl)ethyl, 2-methylthioethyl, tert-butyl, 155839.doc -24- 201204720 3-methyl-3-pentyl, dicyclopropyl fluorenyl , cyclopentyl, cyclohexyl, allyl, decylallyl, cinnamyl, phenyl, decyl, benzyl and substituted benzyl, 2,6-dialkyl phenyl, such as Pentafluorophenyl, 2,6-dialkylphenyl. A preferred protecting group for the acid is an oxime ester or an ethyl ester. Methods of adding (generally referred to as "protection") and removal (generally referred to as "removal of protecting groups") of such amines and acid protecting groups are well known in the art and may be, for example, in PjKocienski , Pr〇tecting Gr〇ups,

Thieme, 1994 (which is incorporated herein by reference in its entirety) and

Gr generation neAWuts, Protective Groups in Organic Synthesis, 3rd (John Wiley & Sons, New York, 1999) is known. Unless otherwise stated, structures described herein are also intended to include all isomeric (e.g., enantiomeric, diastereomeric, and geometric (or conformable) forms) of the structure; For example, the 11 and 8 configurations of each asymmetric center, as well as the double bond isomers and the (Z) and (E) configuration isomers. Thus, single stereochemical isomers and enantiomers, diastereomers, and geometric isomers (or conformable isomers) mixtures of the present compounds are within the scope of this month. Unless otherwise stated, all tautomeric forms of the compounds of the invention are within the context of the invention. For example, a compound of formula t can exist as a tautomeric form as follows:

In addition, 'unless otherwise stated', the structures described herein are also intended to include 155839.doc •25·201204720 The only difference is that in the presence of one or more isotopically enriched core compounds, in addition to hydrogen replaced by hydrazine or hydrazine, or by or UC thickening -. Compounds having the structure of the present invention, as exemplified, are within the replacement carbons of the present invention. *1^* ^ Compounds are suitable for use in bioassays as, for example, analytical tools or probes. ^ Examples of suitable solvents are (but not limited to) water, sterol, dichloromethyl (DCM), acetonitrile, dimethylformamidine. Amine (DMF), ethyl acetate (as a core), isopropanol (IPA), isopropyl acetate (ipac), tetrahydrogen. Tf (thf), methyl ethyl (MEK), tert-butanol and N-methyl biloprofen (nmP). II. Compounds of the Invention In one aspect the invention relates to a pharmaceutical composition comprising a compound of formula I and a compound of formula II and/or a hydrazine compound.

Formula II

155839.doc -26- 201204720

II.A.1. Examples of compounds of formula I In one aspect, the compositions of the invention comprise a compound of formula I,

Or a pharmaceutically acceptable salt thereof, wherein: Ring A is selected from the group consisting of:

(b) (c) (d) 5 (8) R1 is -CF3, -CN or -OCCH2N(CH3)2; R2 is hydrogen, -CH3, -CF3, -OH or -CH2OH; R3 is hydrogen, -CH3, - OCH3 or -CN; the restriction condition is that R2 and R3 are not hydrogen at the same time; Ά In one embodiment, the ring of formula I is (8). 155839.doc -27. 201204720 In an embodiment, ring A of formula I is

(b) In another embodiment, ring A of formula I is 〇

〇

X In another embodiment, ring A of formula I is (6). In one embodiment, Formula I is considered to be a meaning. In another embodiment, R 式 of Formula I is _CN. In another embodiment, ri of Formula I is _C=CCH2N(CH3)2. In one embodiment, R2a_CH3 of Formula I. In another embodiment, R2 of Formula I is {匕. In another embodiment, the rule 2 of Formula I is _011. In another embodiment, R2 of formula I is ΤΗβΗ. In one embodiment, r34_CH3 of Formula I. In one embodiment, r3 of Formula I is _〇cH3. In another embodiment, R3 of Formula I is _CN. In one embodiment, formula iiR2 is hydrogen; and formula jiR3 is _CH3, _〇CH3 or -CN. In another embodiment, R2 of formula I is -CH3, -CF3, -OH or -CH2OH; and R3 of formula I is hydrogen beta 155839.doc -28-, 4 201204720 In several embodiments of the invention, ^ -CF3, R2 is hydrogen; and ruler 3 is _CH3, _〇c only. , R1 is in the example where 'R1 is -CN. In other embodiments in other implementations (CH3)2. In the embodiment, R3 is . For 'CcCCHa or R3 is -CN. 5, R3 is -〇CH3. The other implementation of CF Diming's ring A is V, ra 'f3, R is -CH3, _CF3, _oh or _CH2〇h, and R is in his application, R, -CNe in other real' 'I, wind. In its

(called 2. In the embodiment, r2rh R = cch2N, λ has R 4-cf3 〇吱 R is -OH. Or, R2 is · CH2〇He

In some embodiments of the invention, the ring VIII of the formula I is ν Ά, Rl A AR3^.CH3 〇 cH3^ is _ci^ In other embodiments, R1 is -CsCCH2N (CH3h. In the embodiment, R3 is _〇Ch3. Or, R, _CH" or, r3 is -CN 〇 In other embodiments of the invention, ring A is , and rule 1 of formula 1 is • CF3 9 A. n\j — von-CH3, · <: ρ3, _〇H or _CH2〇H, and rule 3 is hydrogen in its example, and R is -CN. In other embodiments, R丨 is -CsCCH2N (H3)2 〇 In the embodiment, the ruler 2 is _cH3. Or, r2 is · Cf3. Or 'R2 is the collar. Or, R2 is _ch2oh» 155839. (j〇c • 29- 201204720 In several embodiments of the present invention, Ring I of I is I =, with hydrogen; and even, commission 3 or, in other examples, R is -CN. In other embodiments, Rl , , τ κ is -〇cch2n (ch3)2 In the embodiment, r, -CH" or, or, R3 is -CN. -0CH3.

AH In other embodiments of the invention, ring n - cf3 ' R, _CH3, _CF3, _ 〇 H (CH2 〇 H, and R basin I R is _CN of formula I. In other embodiments,

(CH3) 2. In the implementation of money, r2 is C-CCH2N ^ t β I R is -CFi. Where R is -OH. Or, R2 is ·〇Η2〇Η. 〆

pyOH In several embodiments of the invention, the ring -CF3, R2 of formula 1 is hydrogen; and R3A_CH3, _ - in the example, R 丨 helmet 3 4 CN. In other embodiments R is -CN. In other real ΎΡΗ^. τ κ is -c=cch2n (ch3)2. In the implementation of fiscal, r3 is even or R3 is _CI^ and R is -〇CH3.

ilyOH In other embodiments of the invention, the formula CF3, R2 is CH 4 X ′ R1 is ^ . ’’ 3 3 , paved or-called (10), and the R3A household is exemplified. In other embodiments, the second is 虱. In it (in the embodiment, R, _CH ^C_ is the second, R2 is _CF3. or 155839.doc • 30. 201204720, R2 is -OH. Or, R2 is -CH2OH. Representative I The compounds are described in Table 1-1 below. Table 1-1 1 1-2 1-3 She i F 1-4 1-5 1-6 % "Ί> % 1-7 1-8 1-9 % F F 1-10 1-11 1-12 % Scale 1-13 1-14 155839.doc -31 - 201204720 II.A.2. Compound 1 In another embodiment, the compound of formula I is compound 1, known Its chemical name is N-(4-(7-azabicyclo[2.2.1]hept-7-yl)-2-(trifluoromethyl)phenyl)-4-yloxy-5-(trifluoro Methyl)-1,4-dihydroquinoline-3-carboxamide.

NH

F F

N Compound 1 II.A.3. Synthesis of a compound of formula I II.A.3.a. - General procedure Preparation of a compound of formula I Scheme 1-1 : r3

,nh2 H/Hal 1a a

,Hal [I Ί : A o2n

HN. Ri 2a 3

0

OR H/Hal 〇 OR 1b

02N 1. b . 1. b 2. c A 2' d 3. d 3. c R3

0 0

155839.doc -32- 201204720 a) (C02R)2CH=CH(0R), toluene, heating; b) Dowther or Diphenyl ether, reflux, n2 atmosphere; c) removal of halogens that may be present Blocking group (eg -Cl), Pd/C, H2, EtOH; d) removal of protecting group R by base or acid; e) CH3CN, Et3N, heating; f) Pd/C, H2, EtOH; g) HATU , Et3N, DMF or propylphosphonic acid cyclic anhydride (T3P®), "biter than 2-methyltetrahydro". Scheme 1-1 depicts the collection pathway for the preparation of compounds from the substituted benzene derivatives 13 and 23. In the final transformation, bismuth hexafluorophosphate (7-azabenzotriazol-1-yl)-tert-tetradecyl hydrazine (HATU) and triethylamine can be used in N,N-dimethylamine In (DMF), or by using propylsulfonic acid cyclic anhydride (T3P8) and 0-pyridine in 2-mercaptotetrahydrofuran, a compound of formula I is obtained by coupling a carboxylic acid ld with an amine 2c to form a guanamine. From the corresponding substituted benzene derivative la, via the reaction of la with the appropriate malonate ((: 〇 211) 2 (: 11 = (: 〇 11) (where R is alkyl or aryl 'such as Thermally mediated condensation of mercapto, ethyl, tert-butyl, phenyl, p-nitrophenyl or the like to give the starting sequence of jb to prepare the carboxylic acid Id. Compound 1 b via a three-step sequence Conversion to carboxylic acid hydrazone d, the three-step sequence includes intramolecular cyclization (step b) when heated under reflux in Dow heat exchanger or diphenyl ether, followed by removal under catalyzed detoxification conditions (required) a halogen-based barrier group (step c) and an acid or base-catalyzed interest (step #. The order of removal of the protecting group and the saponification step may be reversed; that is, as depicted in the scheme, step c may be in step d Before or after. Referring to Scheme 1-1, the aniline derivative 2c can be prepared from the nitrobenzene 23 via a three-step sequence. Thus, in the presence of triethylamine, the nitrobenzene is 5% and the 155839.doc-33 - 201204720 • The coupling of the cyclic amine '·Α”3 as defined in the text gives compound 2b. Palladium catalysis of 2b Original, the process to obtain the amine 2c 1-2: Preparation of a compound of formula I

a) DMSO 'K2C03, 80 ° C; b) N,N-dimethylprop-2-yn-1-amine, Pd(PPh3)2Cl2, Cul, DMF, TEA, 80. (: ; c) Fe, FeS04, H20 or Zn, AcOH, H20; d) HATU, Et3N, DMF or propylphosphonic acid cyclic anhydride (T3P®) 'pyridine, 2-methyltetrahydrofuran. Scheme 1-2 depicts the synthesis of a compound of formula I having a propargylamine side chain. Therefore, nitrobenzene 2a (where Hal is bromine, gas or the like) is as described herein,

HN^z 3 defined by ί a J was coupled in DMSO in the presence of potassium carbonate to give Intermediate 4. The intermediate 4 is coupled with ruthenium, ruthenium dimethylpropan-2-alkyne-i-amine by palladium catalysis 155839.doc -34 * 201204720, followed by iron or zinc catalyzed reduction of the nitro moiety to give the amine 5 . Coupling of amine 5 with carboxylic acid Id gives 6. Scheme 1-3: Preparation of some compounds of formula j

R, 8

a) DMSO ' K2C03 ' heating, or CH3CN, TEA ' heating; b) PGX (such as TBDMSC1), test (such as imidazole), DMF; c) h2,

Pd/C, EtOH; d) HATU, Et3N, DMF or propylphosphonic acid cyclic anhydride (T3P®), pyridine '2-methyltetrahydrofuran; e) removal of PG' such as HC1,

EtOH. PG = protecting group; X = leaving group. ~ ^ / \ » a * Scheme 1-3 depicts the synthesis of a compound of formula I, wherein -3 is a 7-azabicyclo[2·2·1]g having an external hydroxyl group or an internal hydroxyl group at the 2-position. alkyl. Adduct substituted with 155839.doc -35- 201204720 base (+)_内-7_azabicyclo[2.2.1]heptan-2-ol, (-)-end-7-azabicyclo[2.2 .1]heptan-2-ol, (+)-external-7·azabicyclo[2.2.1]heptan-2-ol and (i)-exo-7-azabicyclo[2.2.1]heptan-2-ol Can be used, for example, Fletcher, SR et al., "Total Synthesis and Determination of the

Absolute Configuration of Epibatidine, j. C/zew, 59 pp. 1771-1778 (i994). 7_Azabicyclo[2.2.1] Geng itself is commercially available from Tyger Scientific inc. 324 Stokes Avenue Ewing, NJ, 08638 USA. Thus, as in one of the series of transformations outlined in Schemes 1 _ 1 and 1 _2, compound 2a is coupled with a bicyclic [2.2. oxime] amine of formula 7 to provide a compound of formula 8. If the compound of formula 8 has a hydroxyl group, then in step b it may be necessary to protect the hydroxyl group with a protecting group such as a decyl protecting group prior to subsequent conversion. Treatment of the alkylated compound of formula 8 with a known condition ', such as gasified tributyl decyl decane, affords the protected compound of formula 9. Reduction of the nitro moiety provides the amine of formula 10. Formation of the indoleamine with Id (see Scheme and removal of the hydroxy protecting group (if required, step e) gives the compound of formula 11 which is also a compound of formula. ΙΙ·Α·4·Example: Synthesis of compound i Intermediate 1 : 4- Sideoxy-5-(trifluoromethyl)_;!,4·dihydroquinoline-3carboxylic acid (17)

155839.doc -36 - 201204720

OEt 5 M NaOH eot

10% Pd/C, H2l Et3N

EtOH, 5X: > 90% Example la: 2-((2-Ga-5-(trifluoromethyl)anilino)methylene)malonate (14) in a nitrogen atmosphere 2-Chloro-5-(trifluoromethyl)aniline 12 (200 g, 1.023 mol), 2-(2-) in a three-neck 1 L round bottom flask equipped with a Dean-Stark condenser. Ethoxy acetamido) diethyl malonate 13 (276 g '1.3 mol) and toluene (1 〇〇 mL). The solution was heated to 140 ° C with stirring and maintained at this temperature for 4 hours. The reaction mixture was cooled to 70 ° C and hexane (600 mL) was slowly added. The resulting slurry was mixed and warmed to room temperature. The solid was collected by filtration, washed with hexane (2×4 〇〇 mL) containing 1% EtOAc, and then dried under vacuum to give the desired condensation product 2-((2- s- 5-(3) A white solid of fluoromethyl)anilino)methylene)malonate 14 (350 g, 94% yield). NMR (400 MHz, DMSO-d6) δ 11.28 (d, /=13.0 Hz, 1H), 8.63 (d, /=13.0 Hz, 1H), 8.10 (s,1H), 7.80 (d, *7=8.3 Hz , 1H), 7.50 (dd, "7=1.5, 8.4 Hz, 1H), 4.24 (q, "/=7.1 Hz, 2H), 4.17 (q, "7=7.1 Hz, 2H), 1.27 (m, 6H) ).

Ethyl Formate (15) Method 1 D〇wther_ (200 mL, 8 mL/g) was fed into an L flask and degassed for 1 hour at 2 °C. The solvent was heated to 26 Torr. And fed 2-((2-a-5-(trifluoromethyl)anilinyl)methylene)malonate 14 (25 g) in portions by 〇 155839.doc •37- 201204720 , 0.07 mol) 〇 at 260. (The mixture obtained was stirred for 6 hours (h) and the obtained ethanol by-product was removed by distillation. The mixture was slowly cooled to 80 ° C. Hexane (15 mL) was slowly added over 30 minutes (min), and then 200 mL of hexane was added in one portion. The slurry was stirred until it reached room temperature. The filtered solid was washed with hexane (3×150 mL) and then dried under vacuum to give 8- gas-4 as a tan solid. -Phenoxy-5-(trifluoromethyl)-I, 4-dihydroindan-3-carboxylate 15 (13.9 g '65% yield). Nmr (400 MHz, DMSO-d6) δ 11.91 ( s&gt; 1H), 8.39 (s, 1H), 8.06 (d, /-8.3 Hz, 1H), 7.81 (d, 7=8.4 Hz, 1H), 4.24 (q, /=7.1 Hz, 2H), 1.29 ( t, "7 = 7.1 Hz, 3H." Method 2 Compound 14 (2000 g, 5.468 mol) was introduced into the reactor. Dow heat conductor (4.000 L) was fed into the reactor at room temperature. Nitrogen purge to degas it overnight. Then it was stirred and warmed to 26 Torr to distill off the resulting EtOH. The reaction was monitored and the reaction was completed after 5 5 hours. The heat source was removed and the reaction mixture was cooled to 8 Torr. Feed heptane (2.000 L). Stirring and mixing 30 minutes. Feed the heptane (6.000 L) to the stirred mixture and continue stirring overnight. The solid was filtered off and washed with heptane (4 〇〇〇L) and in a vacuum oven at 5 ° C Drying underneath gave compound 15. Example lc: 4-Alkyloxy-5-(trifluoromethylquinoline-3-carboxylic acid ethyl ester (16) 8-ox-4-oxo-5-(trifluoromethyl) ^, dehydroquinoline _3_carboxylic acid ethyl ester 15 (100 g, 0.3 mol), ethanol (125 〇 mL, 12 5 mL / g) and triethyl 155839.doc • 38- 201204720 amine (220 mL, 1.6 mol) was fed into a 3-neck 5 L flask. Then 10 g of 10% Pd/C (5 〇./. wet) was fed into the vessel at 5 ° C. Stirring vigorously under hydrogen atmosphere at 5 ° C The reaction was allowed to react for 20 hours, after which time the reaction mixture was concentrated to a volume of about 15 〇mL. The product was obtained as a Pd/C-containing slurry, 4- oxo-5-(trifluoromethyl)-1 quinone-quinoline-3- Ethyl formate 16 was used directly in the next step. Example Id: 4-Phenoxy-5-(trifluoromethyl)-1,4-dihydroquinoline-3-carboxylic acid (17) with reflux condenser In a 1 L flask, 4-sided oxy-5-(trifluoromethyl)-1 Η-islan-3-ethyl oleate 16 (58 g, 0.2 mol, containing pd/C thick The reaction slurry) was suspended in NaOH (814 mL, 5 Torr, 4.1 mol) and heated at 80 C for 18 hours' and then heated at 1 °C for an additional 5 hours. The reaction was warmed by filling the diatomaceous earth to remove Pd/C and the celite was washed with 1 N NaOH. The filtrate was acidified to about pH 1 to give a thick white precipitate. The precipitate was filtered&apos; followed by rinsing with water and cold acetonitrile. The solid was then dried <RTI ID=0.0></RTI> to <RTI ID=0.0></RTI> to <RTI ID=0.0></RTI> <RTIgt; 〇〇〇MHz, DMSO-d6) δ 15.26 (s, 1H), 13.66 (s, 1H), 8.98 (s, 1H), 8.13 (dd, *7=1.6, 7.8 Hz, 1H), 8.06-7.99 ( m, 2H). Alternative preparation of intermediate 1: 4·sideoxy_S·(trifluorodecyldihydroquinolin-3-indoleic acid (17)

Example le: 8-gas-4-oxo-5-(trifluoromethyl)dihydroquinoline _3 155839.doc -39- 201204720 Formic acid (15a) 8-chloro-4-o-oxyl_5 -(dioxamethyl)_1,4_diazepinecarboxylic acid ethyl ester (15) (1200 g, 3.754 mol) was fed into the reaction vessel, followed by 2 -propanol (1.200 L) and water (7.200 L) and Stir. Sodium hydroxide (6 〇〇 6 g ' 7.508 mol) and water (1.200 L) were mixed and cooled to room temperature. The resulting mixture was fed into a reaction vessel and then heated to 80. The mixture was stirred for 3.5 hours to give a dark homogeneous mixture. After another hour, acetic acid (9-599 L' 20% w/v' 31.97 mol) was added via a dropping funnel over 4 5 minutes. The reaction mixture was cooled to 22 ° C at a rate of 6 ° C / hour under a drop. The resulting solid was washed with water (3 L) to give a wet cake (yield: 436 g). The filtrate was dried with Drierite® under a nitrogen bleed in vacuo to give a brown solid. Gas-4-oxooxy-5-(trifluoromethyl)·ι,4-dihydroquinoline_3_carboxylic acid (1069 g). The 8-vapor-4-oxo-5-(trifluoromethyl)-I,4-dihydroquinoline-3carboxylic acid was purified by slurrying in 1.5 L of methanol and stirring for 6 hours. Then it was filtered and dried to give 968.8 g of purified 8- gas-4-oxooxy-5(trifluoromethyl)-1, 4-dihydroquinolin-3-carboxylic acid. Example If: 4-Phenoxy-5-(trifluoromethyl)-M•dihydroquinoline-3carboxylic acid (17) Compound 15a (18.5 g, 1.00 equivalent, limited reagent) was fed into the reaction vessel, and Me〇H (U8 mI^, 64 volumes) was added under stirring in an inert atmosphere. Sodium methoxide (3.53 g, 丨.00 eq.) was added portionwise to the reactor over 10 minutes. Stir the mixture until all solids have dissolved (5 to 1 〇 minutes). Palladium on carbon (2.7 g, 0.03 equivalents) was then added to the reaction mixture. The formic acid I55839.doc -40 - 201204720 (10.78 g, 2 equivalents) dissolved in MeOH (67 mL, 3.6 vol) was added to the reaction mixture over 30 minutes [or the potassium formate reagent was changed to hydrogen] . Then, it is stirred at ambient temperature for about 4.5 hours, 1 8-chloro-4·sideoxy·5_(三敦曱基)_ i,4_二气喧琳冬酸 phase, on 4-sideoxy_5_ (Trifluoromethyl)_M•Dihydro-μ”·carboxylic acid ((7) is at most 1.0%, the reaction is judged to be completed. When the reaction is completed, the quality of the earthworm is used. 8_Chloro-cold-oxyl 5 (-fluoromethyl)-丨, 4-dihydroquinoline_3•carboxylic acid in the human container is doubled.) The mixture is filtered to remove the solid. Use ^^〇汛37 mL , 2 volumes) washed diatomaceous earth filter cake. The filtrate was fed into a clean reaction vessel and stirred. Acetic acid (7.22 mL, 2 equivalents) was continuously fed to the stirred solution over a period of at least 45 minutes and the resulting slurry was stirred for 5 hours to 16 hours. The solid was filtered and washed with EtOAc (EtOAc) elute Intervening 2 : 4-(7-Azabicyclo[2.2.1]hept-7-yl)_2(trifluoromethyl)aniline (20)

Ch3cn, tea 80 &quot;C, 16 h

Pd/C, Hz EtOH, 12 h

Example lg. 7-[4-Nitro-3-(trifluoromethyl)phenyl]·7-azabicyclo[2.2.1]heptane (19) Method 1 To accommodate 7-aza in a nitrogen atmosphere Bicyclo [2.2.1] heptane hydrochloride 7a (4.6 g ' 34.43 mmol, from Tyger Scientific Inc., 324 Stokes

Avenue, Ewing, NJ, 〇8638 USA obtained) Add 4_ Fluorine 155839.doc -41 _ 201204720 Nitro-2_(trifluoromethyl)benzene 18 (6.0 g, 28.69 mmol) and triethylamine (8.7 g) , 12.00 mL, 86.07 mmol) in acetonitrile (50 mL). The reaction flask was heated at 80 ° C for 16 hours under a volatile atmosphere. The reaction mixture was cooled and then partitioned between water and dioxane. The organic layer was washed with 1 M EtOAc then dried over Na 2 EtOAc. Purification by gelatin chromatography (0% to 10% ethyl acetate) afforded 7-[4-nitro-3-(trifluoromethyl)phenyl]-7- Heterobicyclo[2.2.1]heptane (19) (7.2 g, 88% yield). NMR (400.0 MHz, DMSO-d6) δ 8.03 (d, J = 9A Hz, 1H), 7.31 (d, J = 2A Hz, 1H), 7.25 (dd, J = 2.6, 9.1 Hz, 1H), 4.59 ( s, 2H), 1.69-1.67 (m, 4H), 1.50 (d, *7=7.0 Hz, 4H). Method 2 4-Fluoro-1-nitro-2-(trifluoromethyl)benzene (18) (901 g, 4.309 mol) was combined with Na2C03 (959.1 g, 9.049 mol) and DMSO under nitrogen atmosphere. 5 L, 5.5 vol.) were introduced together into a 3 〇 jacketed container. Next, 7-azabicyclo[2.2.1]heptane hydrochloride (7a) (633.4 g, 4.740 mol) was added portionwise to the vessel. The temperature is gradually increased to 55. Hey. When the reaction was substantially complete, the mixture was diluted with 10 volumes of EtOAc and washed three times with water (5.5 volume) or until the DMSO disappeared (HPLC) in the aqueous layer. The organic layer was concentrated to 4 volumes, and then the solvent was exchanged with cyclohexane until all of the ones were removed (and the total volume in the flask was about 4 volumes (containing cyclohexane). The reaction mixture was heated to 6 Torr and maintained for 3 Torr. The solution was then cooled to room temperature with stirring or rotation for 3 hours. When all solids were crystallized, the solution was concentrated to dryness to give 7_[4_ Nitro_3_(trifluoroanthracene 155839.doc 42·201204720 base)phenyl]-7-azabicyclo[2.2.1]heptane (19). Method 3 4F 1nitro-2-(difluoromethyl) The benzene (18) is dissolved in 3 volumes. Add tetrabutylammonium bromide (〇〇5 equivalent) and hydrazine (5〇, 3 6 里). The receiver is added (7 to 7t). Azabicyclo[2 2 heptahydrochloride (7a). The reaction was allowed to warm to ambient temperature and was monitored by HpLc. After completion, the layers were separated and washed with 1 M EtOAc. The organic layer was washed once with water, once with brine, and then distilled. The obtained material was recrystallized from cyclohexane under reflux. It was washed with % hexane and dried under a vacuum purge at 45 under vacuum to give 7·[4-nitro-3-(trifluoromethyl)phenyl]-7-azabicyclo[2.2.1 Heptane (19). Example lh: 4-(7-azabicyclo[2.2.1]hept-7(yl)_2_(trifluoromethyl)aniline (20) will be fed with 7-[4-nitrogen Flask of 3-(3-trifluoromethyl)phenyl]-7-azabicyclo[2.2.1]heptane 19 (7.07 g, 24.70 mmol) and 10% Pd/C (0.71 g, 6.64 mmol) Then, it was boiled with nitrogen. Ethanol (22 mL) was added and the reaction flask was charged with a hydrogen balloon. After vigorously stirring for 2 hours, the reaction mixture was purged with nitrogen and Pd/C was removed by filtration. The filtrate was concentrated to a dark oil, and the residue was purified by silica gel chromatography (yield: </RTI> to 5% ethyl acetate in hexane) to afford 4-[7-azabicyclo[ 2.2.1]hept-7-yl)-2-(trifluoromethyl)aniline (20) (5.76 g, 91% yield) NMR (400.0 MHz, DMSO-d6) δ 6.95 (dd, «7= 2.3, 8.8 Hz, 1H), 6.79 (d, J=2.6 Hz, 1H), 6.72 (d, 7=8.8 Hz, 155839.doc •43- 201204720 1H), 4.89 (s, 2H), 4.09 (s, 2H), 1.6 1-1.59 (m, 4H) and 1.35 (d, •7=6.8 Hz, 4H) ° Example li: Preparation of 4-(7-azabicyclo[2.2.1]hept-7-yl)_2_(trifluoromethyl) Aniline hydrochloride (20-HCI)

Cf3 19

Cf3 20-HCI feeds the carbon/carbon (150 g' 5% w/w) into a Btichi Hydrogenator (20 L capacity) under nitrogen, followed by the addition of 7_[4_nitro_3_ ( Dimethyl)amino]7-azabicyclo[2.2.1] heptane (19) hydrochloride (15 00 g) and 2-mercaptotetrahydrofuran (10.5 L'7 vol). Hydrogen is fed into a closed vessel to a pressure above atmospheric pressure + 0.5 bar. A vacuum is applied for about 2 minutes, followed by introduction of hydrogen to a pressure of 0.5 bar. This process is repeated 2 times. Hydrogen is continuously fed at +0.5 bar above atmospheric pressure. The mixture was searched and the temperature was maintained at 18 by means of a jacket for the cooling vessel. 〇 with 23. Between 〇. After the reaction no longer consumes hydrogen and no longer exotherms, a vacuum is applied. Nitrogen gas was fed into the vessel at 05 bar and a vacuum was applied, followed by a nitrogen gas of 05 bar. When the reaction was substantially complete, the reaction mixture was transferred to a receiving flask via a filter funnel under a nitrogen atmosphere using a Shih-Hain filter. The diatomaceous earth filter cake was washed with 2 -methyltetrahydrofuran (3 L, 2 vol). The wash liquor and filtrate are fed into a vessel equipped with agitation, temperature control and a nitrogen atmosphere. A 4 M HCl solution of 1,4-dioxane (1 vol) was continuously added to the vessel over 1 hour at 20 °C. The mixture was stirred for an additional 1 hour (or overnight), filtered and washed with 2 _ _ s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s Heterobicyclo[2.2.1]hept-7-yl)-2_(trifluoromethyl)aniline hydrochloride (20-HC1). Example lj: Preparation of Compound 1

To 4-sided oxy-5-(trifluoromethyl)-1 quinone-quinoline-3-carboxylic acid 17 (9.1 g '35.39 mmol) and 4-(7-azabicyclo[2.2.1] at room temperature Addition of propylphosphonic acid cyclic anhydride to a solution of p-heptyl-7-yl)-2-(difluoroindolyl)aniline 20 (9.2 g, 35.74 mmol) in 2-methyltetrahydrofuran (91.00 mL) T3P, 50% ethyl acetate solution '52.68 mL, 88.48 mmol) and a specific bite (5.6 g, 5.73 mL '70.78 mmol). The reaction flask was heated at 65 ° C for 10 hours under a nitrogen atmosphere. After cooling to room temperature, the reaction was diluted with EtOAc EtOAc EtOAc. The layers were separated and the aqueous layer was extracted twice with ethyl acetate. The combined organic layers were washed with EtOAc EtOAc m. The crude solid product was slurried in ethyl acetate / diethyl ether (2:1), which was collected by vacuum filtration and washed twice with ethyl acetate / diethyl ether (2:1) to give pale yellow crystalline powder. The product of the shape. The powder was dissolved in warm ethyl acetate and absorbed on diatomaceous earth. Purification by gelatin chromatography (0% to 50% ethyl acetate in dioxane) afforded N-(4-(7-azabicyclo[2.2.1]hept-7- Base)-2-(dione fluorenyl) benzyl)-4- oxo-5-(tris-methyl)-1,4-diazepine-3- 155839.doc •45- 201204720 Amine (13. 5 g, 76% yield). LC/MS m/z 496.0 [M+H]+, </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> <RTIgt; 4 NMR (400.0 MHz, DMSO-d6) δ 13.08 (s, 1H), 12.16 (s, 1H), 8.88 (s, 1H), 8.04 (dd, J=2A, 7.4 Hz, 1H), 7.95-7.88 ( m, 3H), 7.22 (dd, 2.5, 8.9 Hz, 1H), 7.16 (d, J=2.5 Hz, 1H), 4.33 (s, 2H), 1_67 (d, /=6.9 Hz, 4H), 1.44 ( d, ·7=6·9 Hz, 4H). Synthesis of 7-azabicyclo[2.2.1]heptane hydrochloride (7a)

OMs C

NaOH aqueous solution, then Η0Ι (gas 1

Nh2 r^s| Na2C03 or K2C03 DCM, Boc20 NHBoc ό NHBoc MsCI, TEA DCM OH 0Η OMs Starting material: trans-4-aminocyclohexanol A B

TFA Example lk: Preparation of trans-4-(t-butoxycarbonylamino)cyclohexanol (A) Method 1 Sodium carbonate (920.2 g, 8.682 mol, 2 eq.) was added to the reaction vessel followed by the addition of water ( 3.000 L, 6 volumes) and stirred. Dioxane (DCM, 4.000 L, 4 vol) was added followed by trans-4-aminocyclohexanol (5 00.0 g, 4.341 mol) to give a two-phase reaction mixture which was stirred vigorously at room temperature. The solution of Boc20 (947.4 g, 997.3 mL, 4.341 mol, 1 eq.) in DCM (2 vol) was then quickly added dropwise to the vessel, and 155839.doc -46 _ 201204720 was stirred at room temperature overnight. . The reaction mixture was then filtered and the cake was washed with water (2×8 vol). The product was suction dried until it was a tight cake. The cake was then dried in a vacuum oven at 35 ° for 24 hours to give 830 g of the reverse (t-butoxycarbonylamino) cyclohexanol (A) as a crystalline solid. Method 2 Two 50 L three-necked round bottom flasks were each equipped with a mechanical stirrer and thermocouple. The flask was placed in a cooling bath, and then water (8 87 L) and trans-4-aminocyclohexanol (1479 g) were fed into each flask. After about 1 to 3 minutes, the anti--4 Aminocyclohexanol was dissolved, and potassium carbonate (1774_6 g) was added to each flask. After about 2 minutes, potassium carbonate was dissolved and DCM (2 96 L) was fed into each flask. Then, Boc anhydride (3082.6 g) in DCM (1479 mL) was added to each flask at a rate to maintain the temperature at 20 °C to 30 °C. Using an ice/water bath to control the amount of heat and accelerate the addition takes about 1 to 2 hours. A suspension was formed during the addition and the reaction mixture was allowed to warm to room temperature and was searched overnight until the reaction was completed based on the disappearance of the Boc anhydride. Heptane (6 L) was then fed into each flask and the mixture was cooled to about 〇. 〇 to 5 C. Solids were collected from each flask by filtration using the same filter. The combined solids were washed with geni (6 L) and water (8 L). The solids are fed into a suitably sized canister equipped with a mechanical stirrer. Water (12 L) and heptane (6 L) were added and the resulting suspension was mechanically removed for 6 min. The solid was collected by tearing, and then washed with water (8 L) and heptane (8 L) on a filter, air dried on a filter for three days' and then dried under vacuum at 3 Torr to 35. Constant weight' gives the product as a white solid. 155839.doc •47·201204720 Example 1 : Preparation of methanesulfonic acid trans-4-(t-butoxycarbonylamino)cyclohexyl ester (B) Method 1 12 L flask was equipped with a nitrogen stream and a mechanical stirrer. Introduce trans-4_(t-butoxycarbonylamino)cyclohexanol (750 g, 3.484 mol), then introduce tetrahydrofuran (THF ' 6.000 L '8 vol) and stir the mixture "addition of triethylamine (370_2 g, 509.9) mL ' 3.658 mol ' 1.05 eq.) and the mixture was cooled to 0 C. While maintaining the temperature of the mixture below 5 °C, carefully add decanesulfonium (419.0 g, 283.1 mL, 3.658 mol, 1.05 equivalent) dropwise. After the addition, the mixture was allowed to stand at 0 C for 3 hours, and then gradually warmed to room temperature (17 C) and mixed overnight (about 15 hours). The mixture was quenched with water (6 vol) and stirred for 15 minutes. Ethyl acetate (Et〇Ac, 9 〇〇〇 L, 12 vol) was added and stirring was continued for 15 minutes. Stirring was stopped and the mixture was allowed to stand for 10 minutes' and the aqueous phase was removed. Add 1 N HCl (6 vol, 4.5 L) and continue mixing for 15 minutes. Stop searching and remove the water phase.丨〇% w/v NaHC〇3 (4.5 L, 6 vol) was added and the mixture was mixed for 1 Torr. Stop mixing and remove the water phase. Water (6 volumes, 4.5 L) was added and the mixture was stirred for 10 minutes. The aqueous layer was removed and the organic layer was carefully filtered and concentrated to 4 volumes. Gengyuan (5.5 vol, 4 L) was added and the mixture was concentrated to dryness to give 988 g of dec. Method 2 A three-necked round bottom flask equipped with a mechanical stirrer, addition funnel, nitrogen inlet, thermocouple and drying tube was placed in a cooling bath. Anti-4·(Tertibutoxycarbonylamino)cyclohexanol (2599 g, 12.07 mob 1.0 equivalent), tetrahydrogen I55839.doc -48·201204720 furan (THF) (20.8 L) and triethylamine (1466 g, 14.49 mo 1.2 equivalent) was added to the flask. The mixture was cooled with an ice water bath and stirred. The ruthenium chloride (1466 g, 12.80 mol, 1.06 equivalent) was added dropwise over 1 hour by means of a feed funnel. After the addition was completed, the cooling bath was removed and the reaction mixture was stirred until TLC indicated the starting material was consumed (about 30 minutes). The reaction mixture was then quenched with aqueous HCl ( EtOAc EtOAc (EtOAc) The mixture was stirred at ambient temperature for about 10 to 20 minutes and then transferred to a separatory funnel. The layers were separated and the aqueous layer was discarded. The organic layer was washed with water (2×4.5 L), EtOAc. The mixture was filtered and the filter cake was washed with EtOAc (2x 600 mL). The combined washings and filtrate were concentrated under reduced pressure at 40 ° C to leave a white solid. The solid was dissolved in heptane (3 L) and cooled in an ice/methanol cooling bath. Additional heptane (5 L)' was added and the mixture was searched at 0 °C to 51: for at least 1 hour. The solid was then collected by filtration, washed with cold heptane (0 ° C to 5 ° C, 2 x 1.3 L), and dried under vacuum to 4 (TC) to constant weight to afford product. The reactor was replaced by a round bottom flask with a cooling bath and an ice bath. Example lm: Preparation of trans-4-aminocyclohexyl methanesulfonate (C) Method 1 Put a decane sulfonic acid under a nitrogen atmosphere and an open vent. Trans-4-(t-butoxycarbonylamino)cyclohexyl ester (985 g, 3.357 mol) was introduced into a 3-neck 12 L flask equipped with a stirrer. DCM (1.970 L, 2 vol) was added at room temperature. And stirring was started. Trifluoroacetic acid (TFA) (2.844 kg, 1.922 L, 24.94 155839.doc -49 - 201204720 mol, 2 volumes) was slowly added to the mixture in two batches (1 L per batch). After the addition, the mixture was stirred for 30 minutes and then added a second time. The mixture was stirred overnight (15 hours) at room temperature to give a clear solution. Then 2 曱 四 四 β ( 4 4 4 4 4 4 4 4 4 Immerse it for 1 hour. The receiver carefully carefully smashed the mixture in a ventilated tree and suctioned it to dryness. Using excess TFA, 1100 g of methane sulfonate was obtained. Acid trans-4-aminocyclohexyl ester TFA salt Method 2 50 L three-necked round bottom flask equipped with a mechanical stirrer, addition funnel and thermocouple, and placed in a cooling bath. Add methanesulfonic acid to the flask. 4_(Tertibutoxylkamino)cyclohexyl ester (3474 g, 1. 〇 equivalent) and dcm (5.9 L) was added to the flask. The resulting suspension was stirred at ambient temperature for 5 to 1 minute. Then, trifluoroacetic acid (TFA, 5.9 L) was slowly added via an addition funnel over 2.5 hours to control the exotherm and gas evolution rate produced. The reaction mixture was stirred overnight at ambient temperature and then cooled to I5C to 2G using an ice water bath. °C. Then add 2·methyltetrahydrofuran (2 mg, n 8 L) via the addition funnel to maintain the internal temperature below the hunger rate (about 1.5 hours). Add the initial 4-5 L 2-MeTHF The product was obtained as a white solid. The product was obtained as a white solid. The product was obtained from the crude oil, and then washed with EtOAc (2×2 2 EtOAc). In: Preparation of 7-azabicyclo[221] heptanoic acid salt (7 hook method 1 decane The TFA salt of sulfonic acid trans-4-aminocyclohexanoic acid (2〇〇g, μ&quot; mm〇l) was introduced into a 3 L 3-neck flask, followed by the addition of water (22〇〇L, 11 bodies 155839.doc 201204720 product While maintaining the temperature of the reaction mixture below 25, NaOH (78.11 g ' 1.953 mob 3 equivalents) was slowly added, and the mixture was stirred overnight. Then DCM (1.4 L, 7 vol) was added and the mixture was stirred and the organic layer was separated. The aqueous layer was then extracted again with DCM (1.4 L, 7 vol) and the DCM layer was combined. Then, HC1 (108.5 mL, 12 μ, 1.3020 m〇l, 2 eq.) was added, and the mixture was stirred for 30 minutes, and then concentrated to dryness on a rotary evaporator. Acetonitrile (10 vol) was added and the mixture was concentrated. This operation was repeated 3 times until all traces of water were removed in an azeotropic manner to give 7-azabicyclo[2.2.1]heptane hydrochloride (7a). The crude product was crystallized from acetonitrile (1 vol.) to afford 7-azabicyclo[2 2 </RTI> </RTI> <RTIgt; WNMR (DMSO-d6) ppm 8·〇2·8 〇4 (d); 7 2 2 1.63-1.75 (m); 7.31 (m); 4.59 (s); 3.31 (s); 2.51-3.3 (m) ; 1.45-1.62 (m). Please note: it can also be around 95t to 97. The crude product was distilled off under the agitation and further recrystallized without the addition of DCM for extraction. Method 2 A 5 〇 L three-necked round bottom flask was equipped with a mechanical stirrer, addition funnel and thermocouple and placed in a heating mantle. Methanesulfonic acid trans-4-aminocyclohexyl ester trifluoroacetate (3 〇〇 g, 1 eq.) in water (3 〇 L) was added to the flask. Stirring: At the same time, the mixture was maintained at a temperature lower than 25 t (because the addition caused a slight exotherm) by adding 5 〇% Na〇H (2343 layers, Μ Μ: '3 equivalents) ^Na〇H by the addition funnel After completion, the reaction mixture was searched at room temperature "Do not overnight. The product was recovered by fractional distillation (vapor head temperature 95C to 98C) at reflux temperature (about (10). By adding the positive value of each error) Adjust 155839.doc -51· 201204720 to 2, and concentrate under reduced pressure at 55 ° C, add acetonitrile (ACN, 1.5 L) and stir the resulting suspension for 3 ,, but to 〇 ° C to 5 ° c And maintain for 1 hour. to 5 ° C) ACN (2 x 600 mL) wash value is heavy. Leave a thick paste. Add the suspension for 30 minutes, and then cool to collect the solid by filtration, with cold (〇.〇 And dried under vacuum at 5 〇t: to a 22 L 3-neck round bottom flask equipped with a mechanical stirrer, thermocouple and condenser and placed in a heating mantle. The collected solid (2382 g), methanol (4 7 q and 2-MeTHF (4.7 L) were added to the flask. The resulting suspension was stirred and heated to reflux (about 65 Torr. The reaction flask was transferred to a cooling bath, The mixture was stirred. Then 2_MeTHF (4.7 L·) was added via an addition funnel over 3 min. The resulting suspension was cooled to (TC to and stirred at this temperature for 3 min. Rc) 2_MeTHF (2 x 6 〇〇 mL) washed ' and then dried under vacuum at 50.5 to constant weight. 12 L two-necked round bottom flask equipped with a mechanical stirrer, thermocouple, nitrogen inlet and condenser Place in a heating mantle. Add the crude product (2〇79 g) and (6·2 L) to the flask. Stir the resulting suspension and heat to reflux (about 82 C) for 30 minutes. Transfer the flask to Cool the tank and slowly cool the suspension to 〇. (: to 5. (:, and maintain this temperature for 丨 hours. Collect solids by filtration, use cold (0 ° c to 5 ° c) acn (3x6 〇〇mL) Wash and dry to a constant weight at 55 ° C under vacuum to obtain the product. ΙΙ·Β.

155839.doc -52- 201204720 II. Β 1. Example of a compound of formula II In one embodiment, in the compound of formula II of the composition, hydrazine is -CH2-, -CH2CH2-, -CF2-, -c (ch3)2- or -c(o)-;

Ri' is Η, Cw aliphatic, halo, CF3, CHF2, CKCw aliphatic); and RD1 or RD2 is ZDR9, wherein: ZD is a bond, CONH, S02NH, S02N (Ci.6 alkyl), CH2NHS02, CH2N(CH3)S〇2, CH2NHCO, coo, so2 or CO; and R9 is H, Ci.6 aliphatic or aryl. II.B.2. Compound 2 In another embodiment, the compound of formula II is compound 2 as described below, also known by the chemical name 3-(6-(1-(2,2-difluorobenzo[ ...[^]dioxol-5-yl)cyclopropanecarbamoylamino)-3-methylpyridine-2-yl)benzoic acid.

Compound 2 II.B.3. Summary of Synthesis of Compound 2 The following Scheme 2 - The compound of Formula 11 as exemplified by Compound 2 can be prepared by coupling an acid chloride moiety to an amine moiety according to la to 2-3. 155839.doc -53- 201204720 Process 2-la: Synthetic Acid Vapor Part 1. Restore

C02H 2. NaOH ρΧ〇ΧΧχ〇Η 1. SOC1, — 2. H20

F^C^CI 1. NaCN 2. H20

NaOH

KOH

FX〇XX^CN SOCl2 Scheme 2-la depicts the preparation of 1-(2,2-difluorobenzo[dnu]dioxol-5-yl)cyclopropane carbonyl gas, which is used in Scheme 3 for preparation The indole linkage of compound 2. The starting material 2,2-difluoro benzo[d][l,3]dioxol-5-carboxylic acid is commercially available from Saltig(R) (a subsidiary of Lanxess Corporation). Reduction of the carboxylic acid moiety of 2 2 difluorobenz[d][l,3]dioxol-5-carboxylic acid to a primary alcohol, followed by the use of sub-sparing Μy Li Ganji 1 酝 gas (SOC12 Converting it to the corresponding vaporizer' to get 5_(chloromethyl)_2,2-difluorobenzo-, and 3]dioxolane, which is converted to 2_(2,2-di) with sodium cyanide Stupid and just, "dioxanthyl" acetonitrile. Treatment of 2_(2 benzo[d]n,3]dioxolane ^ ^ (, 2 with alkali and bromo-2-chloroethane Fluorine #rdln ^ decene _5·yl) acetonitrile to give 1-(2,2-difluorobenzo[][1,3]-oxocyclopentene "Gan, (2- 2-fluoro...lri a cyclopropane carbonitrile. The base is used to convert the 155839.doc •54· 201204720 nitrile portion of 1-(,-gaso-,3,dioxol-5-yl)cyclopropanecarbonitrile to a carboxylic acid to give 1-(2,2-difluorobenzo[d][l,3]dioxanthene-5-yl)cyclopropanecarboxylic acid, which is converted to the desired acid using sulphur Vapor. Process 2-1 b. Alternative Synthesis of Acid Vaporization Section &gt;ca

Pd(dba)2, /-B1J3P ---► Na3P04, toluene, H2O, 70°C

Br + E human CN F^〇Et

CN

Br# ♦

NaOH Bu4NBr

3 N HC1, DMSO, lr 75 °C

1. NaOH 2. HC1

Scheme 2-lb provides an alternative synthesis of the desired acid gasification. The compound 5-bromomethyl 2,2-difluoro-1,3-benzodioxole is coupled with cyanoethyl hexaacetate in the presence of a palladium catalyst to form the corresponding alpha cyanoacetate. Partially brewed into a tickic acid to give a cyanoethyl compound. Alkylation of the cyanoethyl compound with 1-bromo-2-chloroethane in the presence of a base affords the cyanocyclopropyl compound. The cyanocyclopropyl compound is treated with a base to give a carboxylate which is converted to the m acid by treatment with an acid. Then use a chlorinating agent (such as sulfite or its analogue) 155839.doc -55-

201204720 Conversion of carboxylic acid to acid hydride. Scheme 2-2: Synthesis of the amine moiety 1. K2C03, Pd(dppf)Cl2 (HO)2B&gt;^ 2. MsOli aqueous solution 3. Aqueous NaOH solution C02tBu -►

C02tBu gland-hydrogen peroxide phthalic anhydride EtOAc, water

COztBu 1. Ms20, py, MeCN 2. Ethanolamine

COgtBu Scheme 2-2 depicts the preparation of the desired tert-butyl 3-(6-amino-3-mercaptopyridine-2-yl)benzoate, which is in Scheme 3 with 1-(2,2-difluoro Coupling of stupid [d][1,3]dioxol-5-yl)cyclopropanecarbonyl to give compound 2. Palladium-catalyzed coupling of 2-oxa-3-methyl D-pyridyl with 3_(t-butoxycarbonyl)phenyl-fatty acid to give 3-(3-mercaptopyridin-2-yl)benzoic acid Tributyl ester, which is subsequently converted to the desired compound. Scheme 2-3: Formation of 3-(6-(1-(2,2-difluorobenzo[d][l,3]dioxol-5-yl)cyclopropanecarboxamido)_3 ·Methylpyridine-2-alkyl)benzoate

• Acid C〇2H 155839.doc • 56- 201204720 Scheme 2-3 depicts the use of triethylamine and 4-dimethylaminopyridine to make 1_(2,2_diIIbenzo[d][l,3] Coupling of oxepan-5-yl)cyclopropanol with 3-(6-amino-3-methyl.0-but-2-yl)benzoic acid tert-butyl ester, initially obtaining compound 2 The third butyl ester. Treatment of the third butyl ester with an acid such as HCl affords the HCl salt of Compound 2, which is typically a crystalline solid. ΙΙ.Β.4·Example··Synthesis Compound 2

Vitride® (sodium hydrogenated bis(2-decyloxyethoxy)aluminum [or NaA1H2 (OCH2CH2OCH3)2]' 65 wt% in toluene) from Aldrich

Chemicals purchased. 2,2-Difluorobenzodioxole_5_decanoic acid was purchased from Saltigo (a subsidiary of Lanxess Corporation). Example 2a ··(2,2-Difluoro-i,3-benzodioxol-5-yl)-methanol l.Vitride (2 eq.)

PhCH3 (〖0 volume) 2·10°/〇(w/w) NaOH aqueous solution (4 when _) FF 0^^kAC02H [92% yield 2,2-fluoro-i,3-benzene commercially available And dioxetane-5-decanoic acid (1 〇 equivalent) was slurried in toluene (10 vol). To maintain the temperature at "the rate to which it is added via the addition funnel vitride (g) (2 equivalents at the end of the addition will be / the degree of water up to 4G ° C and maintained for 2 hours (8), then at the maintenance temperature at 40 C to 50 Under C, carefully add 1〇% (w/w) Ν3〇Η (4·0 eq.) of the aqueous solution (called) via the addition funnel. After stirring for another 3 minutes (min), separate the layers at 40 C. The phase is cooled to (10), then washed with water (volume: volume), dried (Na.sub.2 s.sub.4), filtered and concentrated to give crude (2,2-difluoro- &lt;RTI ID=0.0&gt; Methyl)-methanol, which was used directly in the next step. 0 155839.doc -57- 201204720 Example 2b: S-gasmethyl-2,2-difluoro-1,3-benzodioxole 1. S0C12 (1.5 eq.) DMAP (0.01 eq.) MTBE (5 vol)

82-100% yield

2. Water (4 vol) (2,2-Difluoro-1,3-benzodioxol-5-yl)-methanol (1 eq.) was dissolved in MTBE (5 vol). A catalytic amount of 4_(n,n-dimethyl)aminopyridine (DMAP) (1 mol%) was added and s〇cl2 (1 2 equivalent) was added via an addition funnel. SOCh was added at a rate to maintain the temperature in the reactor at 15l to 2yc. The temperature was raised to 3 (rc and maintained for hrs, and then cooled to 20 ° C. Water (4 vol) was added via an addition funnel while maintaining the temperature below 30 C. After stirring for an additional 30 minutes, the layers were separated. Add 10% (w/V) aqueous NaOH solution (4.4 vol.). After stirring for 15 to 2 minutes, separate the layers. Then dry (NkSO4) organic phase, filter and concentrate to give crude 5-methyl-2,2 -Difluoro_ι,3-benzodioxole, which was used directly in the next step. Example 2c: (2,2-difluoro 4,3·benzodioxole-5 base )_acetonitrile

NaCN (1.4 equivalents) DMSO (3 volumes)

30-40〇C 2. Water (6 volumes) MTBE (4 volumes) Ρν,〇Ύ^

~95-100% yield~~~^ F Add a solution of 5-gashydrazino-2,2-di-U-benzodioxole (1 equivalent) in DMSO (1.25 vol) To Na(:N(i 4 eq.) in DMSO (3 vol) in a slurry while maintaining the temperature at 3 〇 〇 〇 〇 〇 〇 155 155 155 155 155 155 155 155 155 155 155 155 155 155 155 155 155 155 155 155 155 155 155 155 155 155 155 155 Then water (6 vol) was added followed by decyl tertiary butyl ether (MTBE) (4 vol). After stirring for 30 minutes, the layers were separated. The aqueous layer was extracted with MTBE (1.8 vol). The combined organic layers were dried <RTI ID=0.0>(Na2</RTI> <RTI ID=0.0></RTI> </RTI> <RTI ID=0.0> It is used directly in the next step. 4 NMR (500 MHz, DMSO) δ 7.44 (br s, 1H), 7.43 (d, /=8.4 Hz, 1H), 7.22 (dd, 7=8.2, 1.8 Hz, 1H ), 4.07 (s, 2H). Example 2d: Alternative Synthesis of (2,2-Difluoro-1,3-benzodioxol-5-yl) Ethyl Acetate-Acetonitrile

CN

Pd(dba)2, /‘Bu3P CN Na3P04,

Toluene, H20, 70 〇C The reactor was purged with nitrogen and fed toluene (900 mL). The solvent was degassed by spraying nitrogen for at least 16 hours. Then Na3P04 (15 5.7 g, 949.5 mmol) was fed into the reactor, followed by bis(dibenzylideneacetone)palladium(0) (7.28 g ' 12.66 mmol). » » At 23. (: A 10% w/w butyl butyl hexoxide solution (51.23 g, 25. 32 mmol) was fed over 10 minutes from a nitrogen purged addition funnel. The mixture was stirred for 5 minutes. Add 5-dihydro-2,2-difluoro-1,3-benzodioxole (75 g, 316 5 mmol) over a period of 丨. After stirring for another 50 minutes, feed the mixture over 5 minutes. Ethyl cyanoacetate (71.6 g, 633.0 mmol) was then taken in water (4 5 mL). The mixture was heated to 70 over 40 minutes. (:, and the reactants were analyzed by HpLc every 1 to 2 hours. Percentage of conversion. After complete conversion was observed (passing 155839.doc 59·201204720 often 100% conversion after 5 to 8 hours), the mixture was cooled to 2 (rc to 25 °C and filtered through a pad of diatomaceous earth. The earthworms were rinsed with toluene (2 x 450 mL) and the combined organics were concentrated to 300 mL under vacuum at 60 ° C to 65 ° C. DMSO (225 mL) was fed to the concentrate and vacuum Concentrate at 70 ° C to 80 ° C until the effective distillation of the solvent is stopped. Cool the solution to 20 ° C to 25 ° C and dilute to 900 mL with DMSO to prepare for step 2. 4 NMR (500 MHz, CDC1 3) δ 7.16-7.10 (m, 2H), 7 〇 3 (d, 7 = 8.2 Hz, 1H), 4.63 (s, 1H), 4.19 (m, 2H), 1.23 (tj J=7.i Hz, 3H). Example 2e: (2,2-difluoro-1,3-benzodioxole·5·yl)·acetonitrile substitution synthesis

CN feeds 3 N to the above solution of acetic acid (2,2-difluoro-, 3-benzodioxan-5-yl)-1-ethyl-acetonitrile in DMSO over 20 minutes. HC1 (617.3 mL, 1.85 mol) while maintaining the internal temperature below 4 (rc. The mixture was then heated to 75 over 1 hour. (:, and the percent conversion was analyzed by HpLC per hour. When more than 99% conversion was observed) At the time of the reaction (usually after 5 to 6 hours), the reaction was cooled to 2 〇t: to 25 ° C and extracted with hydrazine (2 χ 525 mL) for a time sufficient to completely separate the phases during extraction. The combined organic extracts were washed with % NaCl (2 x 375 mL). The solution was then transferred to a device suitable for a 1.5 to 2.5 Torr vacuum evaporation vessel equipped with a cooled receiving flask. The solution was concentrated under vacuum at less than 6 (rc) To remove the dissolved 155839.doc -60- 201204720 l. Then at 125. (: to 130 it (oven temperature) and 丨 5 to 2 Torr to make ('2 fluoro 1,3 benzodioxane) Pentene-5-yl)-acetonitrile is treated from the obtained oil. From 5·Moistry 2, and the dioxetane is isolated as a clear oil in the % yield (2, 2- Difluoro-1,3-benzodioxol-5-yl)-acetonitrile (2 steps) and Ηριχ purity 9 μ 5% auc (corresponding to 95/. w/w assay). iH NMR (5〇〇MHz, dms〇) § 7 1H), 7.43 (d, J=8.4 Hz, 1H), 7.22 (dd, J=8.2, 1.8 Hz, 1H), 4.07 (s, 2H). Example 2f (2,2·Difluoro_;i,3_benzodioxol-5)) Cyclopropanone

1-Bromo-2-chloroethane (1.5 equivalents) 50% KOH (5.0 equivalents) Oct4NBr (0.02 eq.)

70 °C 88-100% yield at 7 (TC heating (2,2-difluoro-indole, 3-benzodioxole-5-yl)-acetonitrile (1.0 equivalent), 5 〇wt % KOH aqueous solution (5. 〇 equivalent), i• bromo-2- ethane (1.5 eq.) and Ο (^ ΝΒΓ (0.02 eq.) mixture hr. The reaction mixture was cooled, then treated with MTBE and water. The organic phase was washed with brine. The emollient was removed to give (2,2-di-n-1,3-benzodioxol)-cyclopropanecarbonitrile. iH NMR (500 MHZ, DMSO) δ 7.43 (d /=8.4 Hz, 1H), 7.40 (d, J=1.9 Hz, 1H), 7.30 (dd, /=8.4, 1.9 Hz, 1H), 1.75 (m, 2H), 1·53 (m, 2H) Example 2g · l-(2,2-Difluoro-1,3-benzodioxol-5)-cyclopropanecarboxylic acid 155839.doc • 61 - 201204720 MTBE (10 vol) Dicyclohexyl Amine (Equivalent) 1. 6M NaOH (8 equivalents) Ugly 1〇11 (5 volumes), 80°〇

2. MTBE (10 艟祛) 3. MTBE (10 vol) 10% aqueous citric acid solution (8 vol) 69% yield at 80 ° C using 6 M NaOH (8 eq.) in ethanol (5 vol) solution ( 2,2-Difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarbonitrile was hydrolyzed overnight. The mixture was cooled to room temperature and the ethanol was evaporated under vacuum. The residue was dissolved in water and MTBE, 1 μ of HCl was added, and the layers were separated. The ruthenium layer was then treated with dicyclohexylamine (DCHA) (0.97 equivalents). The slurry was cooled to o ° c, filtered and washed with heptane to give the corresponding 1:) (: 11 human salt. The salt was dissolved in MTBE and 10% citric acid and stirred until all solids were dissolved to separate the layers and water The MTBE layer was washed with brine and the solvent was exchanged to heptane, which was then filtered and dried in a vacuum oven at 50. After drying overnight, 1-(2,2-fluoro-1,3-benzodioxane was obtained. Pentene _5_yl)-cyclopropanecarboxylic acid.

(m, 2H), 1.17 (m, 2H) 〇

Alkyl carbonyl

1-(2,2-Difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarboxylic acid 155839.doc • 62- 201204720 (1.2 equivalents) in toluene (2_5 volume) The slurry was made and the mixture was heated to 6 °C. S0C12 (1 4 equivalents) was added via an addition funnel. After 3 minutes, toluene and soci2 were distilled from the reaction mixture. Additional hydrazine (2.5 vol.) was added and the resulting mixture was re-distilled to leave the product acid acid as an oil which was used without further purification. Example 2i · 3-(3-methyl"bite-2-yl)benzoic acid third

(ho)2b^^ C〇2tBu 1. 曱 stupid, 2MK2C〇3 Pd(dppf)a2, 80°C 2·MsOH aqueous solution 3. NaOHi 滋^

COgtBu 2-bromo-3-mercaptopyridine (1.o equivalent) was dissolved in toluene (12 vol). K2C〇3 (4.8 equivalents) was added followed by the addition of water (3.5 volume). The resulting mixture was heated to 65 ° C under turbulent flow for 1 hour. Next, add 3_(tactinoxy Ik group), a base S-ponic acid (1.05 equivalent) and hydrazine (1 ((1卩卩£)〇; 12-(^112〇12(〇.015 equivalent)), and The mixture was heated to 8 Torr. After 2 hours, the heating was turned off, water (3.5 vol) was added, and the layers were separated. The organic phase was washed with water (35 vol.) and 10% aqueous methanesulfonic acid (2 eq. Extraction. The aqueous phase was made up with 50% aqueous NaOH (2 eq.) and extracted with EtOAc (8 vol.). The organic layer was concentrated to give crude 3-(3-methylpyramine-2-yl)phenylhydrazine. The acid tert-butyl ester (82%) was used directly in the next step. Example 2j: 2_(3-(Tertidinoxycarbonyl)phenyl)_3-methylβ-pyridinium oxide

Urea-hydrogen peroxide phthalate liver EtQAc, water C〇2tBu C〇2tBu 155839.doc • 63 · 201204720 3-(3-methylacridin-2-yl)benzoic acid tert-butyl ester (1.0 equivalents) ) Dissolved in EtOAc (6 vol). Water (〇.3 vol) was added followed by urea-hydrogen peroxide (3 equivalents). O-phthalic anhydride (3 equivalents) was then added portionwise to the mixture as a solid at a rate to maintain the temperature in the reactor below 45 °C. After the addition of phthalic anhydride was completed, the mixture was heated to 45 °C. After stirring for an additional 4 hours, the heating was turned off. 1 〇〇 / ❶ w / w Na2S03 aqueous solution (1.5 eq.) was added via an addition funnel. After the NaaSO3 addition was completed, the mixture was further stirred for 30 minutes and the layers were separated. The organic layer was mixed and a wt/wt aqueous Na.sub.2 C.sub.3 solution (2 eq.) was added. After stirring for 30 minutes, the layers were separated. The organic phase was washed with a 13% w/v NaCl aqueous solution. The organic phase was then filtered and concentrated to give crude <RTI ID=0.0>#</RTI> </RTI> <RTIgt; </RTI> <RTIgt; </RTI> <RTIgt; </RTI> <RTIgt; </RTI> <RTIgt; </RTI> <RTIgt; Example 2k: 3-(6-Amino-3-methylpyridin-2-yl)benzoic acid tert-butyl ester

1_Ms2〇, py, MeCN, 70°C 2. Ethanolamine C〇2tBu 2_(3_(Tertibutoxycarbonyl)phenyl)-3-methyl"pyridin-1-oxide (1 equivalent) and A solution of pyridine (4 equivalents) in acetonitrile (8 vol) was heated to 7 〇e &gt; The decane sulfonic anhydride (15 equivalents) was added to MeCN (2 vol.) via an addition funnel over 50 minutes while maintaining the temperature below the crater. The mixture was stirred for a further 0.5 hours after complete addition. The mixture is then cooled to ambient temperature. Ethanolamine (1 〇 equivalent) was added via an addition funnel. After stirring for 2 hours, water (6 vol) was added and the mixture was cooled to hydrazine. After stirring for 3 hours, the solids were collected and washed with water (3 vol), 2:1 acetonitrile/water volume and B. 155839.doc • 64 - 201204720 (2 x 1.5 vol). The solid was dried to a constant weight (&lt;1% difference) in a vacuum oven at 50 ° C under a slight N 2 purge to give 3-(6-amino-3-methylpyridine as a red-yellow solid. 2-yl) benzoic acid tert-butyl ester (53% yield). Example 2: 3-(6-(1-(2,2-difluorobenzo[d][13]dioxol-5-yl)·cyclopropanthene)A-3- Base" than bit-2-yl)-tert-butyl benzoate

The crude acid gasification described above was dissolved in toluene (25 volumes in terms of acid gasification) and added to the 3-6-amino-3_indolylpyridinium-2-ylbenzoic acid via an addition funnel. Third butyl ester (1 eq.), DMAP (0.02 eq.) and triethylamine (3.0 ct. straight) in sputum (3-(6-amino-3 曱 〇 〇 _2 _2 _2 _2 _2) The mixture of tert-butyl citrate in 4 volumes). After 2 hours, water (4 volumes) based on tributyl ethane 6-amino-3-methylpyridin-2-ylbenzoate was added to the reaction mixture. After stirring for 30 minutes, the layers were separated. The organic phase is then filtered and concentrated to give 3-(6-(1-(2,2-dioxabenzo[d][1,3]dioxol-5-yl)cyclopropanoguanidino) A thick oil of -3-mercaptopyridine-2-yl)-tert-butyl citrate (quantitative crude yield). Acetonitrile (3 volumes based on the crude product) was added and distilled until crystallization occurred. Water (2 volumes in terms of crude product) was added and the mixture was stirred for 2 hours. The solid was collected by filtration, washed with 1:1 (by volume) acetonitrile / water (2 x 1 volume as a crude product) and partially dried under vacuum on a filter. The solid was dried in a vacuum oven at 6 °t under a slight...purge: to constant weight (&lt;1% difference)' to give the difluoride as a brown solid 155839.doc -65 - 201204720 benzo[d] [l,3]dioxol-5-yl)cyclopropanecarbamoylamino)-3-methylpyridin-2-yl)-benzoic acid tert-butyl ester. Example 2m: 3-(6-(1-(2,2-difluorobenzo[d][l,3]dioxol-5-yl)cyclopropanecarbamoylmethylpyridine-2 -yl)benzoic acid·Η(:1 salt

To 3-(6-(1-(2,2-difluorobenzo[d][l,3]dioxol-5-yl)cyclopropanecarbamoyl)-3-f-ylpyridine _2_yl)-tert-butyl benzoate (丨.0 equivalent) Water (ο volume) was added to the slurry in MeCN (3.0 volume), followed by the addition of an aqueous solution of agricultural HC1 (0.83 volume). The mixture was heated to 45 ° c ± 5 ° c. After 24 to 48 hours of mixing, the reaction was completed and the mixture was cooled to ambient temperature. Water (1.3 3 volumes) was added and the mixture was stirred. The solid was collected by filtration, washed with water (2 x 0.3 vol.) and partially dried under vacuum on a filter. The solid was dried to a constant weight (&lt;1% difference) in a vacuum oven at 60 ° C under a slight N 2 purge to afford 3_(6·(1(2,2-difluorobenzo) as an off-white solid. [d][l,3]dioxole-5-yl)cyclopropanecarbamoylamino)_3_methylpyridine-2-yl)benzoic acid·Η(:1 salt. Table 2-1 below The physical data of Compound 2 is described. 155839.doc -66- 201204720 Table 2-1 Compounds. Orphan repair uc/Mi 'ofc/RT· 'Life clock N!MQR. ' V 4: Compound 2 453.3 1.93 !HNMR ( 400 MHz, DMSO-d6) 9.14 (s, 1H), 7.99-7.93 (m, 3H), 7.80-7.78 (m, 1H), 7.74-7.72 (m, 1H), 7.60-7.55 (m, 2H), 7.41-7.33 (m, 2H), 2.24 (s, 3H), 1.53-1.51 (m, 2H), 1.19-1.17 (m, 2H). II.C.

II.C.1. Examples of compounds of formula III In one embodiment, in the formula ΙΠ compound: R is Η, OH, OCH3, or two R together form -0CH20- or -ocf2o-; R4 is ruthenium or Alkyl; R5 is hydrazine or F; R6 is hydrazine or CN; R7 is hydrazine, -CH2CH(OH)CH2OH, -CH2CH2N+(CH3)3 or -CH2CH2OH; 118 is 11, OH, -CH2CH(OH)CH2OH, - CH2OH, or feet 7 and 118 together form a five-membered ring. II.C.2. Compound 3 In another embodiment, the compound of formula III is compound 3, known as its chemical 155839.doc -67· 201204720, the name is (i?)-l-(2,2-difluorobenzene And [d][l,3]dioxol-5-yl)-N-(l-(2,3-dihydroxypropyl)-6-fluoro-2-(1-hydroxy-2- Methylpropan-2-yl)-1Η-indol-5-yl)cyclopropanoinamide

Compound 3 X:] II.C.3. Summary of Synthesis of Compound 3 Compound 3 can be prepared by coupling an acid gasification moiety to an amine moiety according to the following scheme. II.C.3.a. Synthesis of acid moiety of compound 3 Acid chloride can be used according to Scheme 2-la, Scheme 2-lb and Examples 2a-2h

The acid moiety of Compound 3 is synthesized in the form of α. II.C.3.b. Synthesis of the amine moiety of compound 3 Scheme 3-1: Synthesis of the amine moiety

TMS

Concentrated HCI room temperature

TMS

CI I.Mg, THF

2. BOMCI TMS

OBn KOH^ MeOH , 0Bn Λ

NBS ” XX^OBn Zn(C〇4)r2H2〇 NH2 2) H2, Pt(S)/C 3)Ts0H*H20 nh2 Et0Ac &lt;0Bn PdtOAcJ.dppb, K2CO3, dtll, water

(MeCN)2PdCI2 © H3N, ^5^Br

155839.doc ** 68 - 201204720 Scheme 3.1 provides an overview of the synthesis of the amine moiety of Compound 3. Conversion of the decyl-protected propargyl alcohol to propargyl chloride, followed by formation of a Grignard reagent and subsequent nucleophilic substitution affords ((2,2-dimethylbutene-3) -Alkynyloxy)methyl)benzene, which is used in another synthetic step. To complete the amine moiety, first bromine the 4-nitrofluoroaniline' and then in a two-step process to convert the anilinoalkyl group with (R)-2-(benzyloxyindenyl)oxirane The initiation begins, followed by reduction of the nitro group to the corresponding amine, which is converted to (7) toluene (4-amino-2-bromo-5-fluoroanilino)_3_(benzyloxy)propan-2-ol The palladium catalyzed coupling of the product with ((2,2·dimethylbut-3-ynyloxy)methyl)benzene (discussed above) affords the intermediate alkynyl compound which is then cyclized to oxime In the oxime portion, the benzyl protected amine moiety of compound 3 is obtained. II.C.3.c. Synthesis of compound 3 by coupling of an acid with an amine moiety. Scheme 3-2. Formation of compound 3

Scheme 3-2 depicts the coupling of an acid to an amine moiety to give compound 3 ^ In the first step, (R)-l-(5-amino(benzyloxy).2_methylpropan-2-yl)_6_ Fluorine oxime 0 wood-1-yl)-3-(present methoxy)propan-2-ol and 1-(2,2-dibenzobenzene 155839.doc • 69· 201204720 = oxygen: ring: "Base" cyclopropane (iv) gas coupling to give benzene benzene. The steps can be carried out in the presence of a base and a solvent. ° 'Alkali' such as triethylamine' and the solvent can be an organic solvent such as DCM or DCM and toluene Mixture. Qi! Home as = step - 'intermediate benzene methylation

: to compound 3. The L step can be completed using reducing conditions sufficient to remove the benzyl group. The reducing conditions may be gasification conditions such as the presence of gas in the presence of a catalyst. II.C.4. Example: Synthesis of Compound 3 II.C.4.a·Amine 3 Partial Synthesis Example 3a: 2-Bromo-5-fluoro-4-nitroaniline

O2n F

NBS νη2

EtOAc 50% 〇2; X5:

Br NH2 was fed to the flask with 3-fluoro-4-nitroaniline (1 〇 equivalent), which was then fed to ethyl acetate (丨〇 volume) and stirred to dissolve all solids. Separately added diammonium imide (1.0 eq.) to maintain an internal temperature of 22 t. At the end of the reaction, the reaction mixture was concentrated on a rotary evaporator in vacuo. The residue was slurried in distilled water (5 vol) to dissolve and remove the succinimide. (The butadiene imine can also be removed by a water treatment procedure.) The water is decanted and the solid is slurried in 2-propanol (5 vol) overnight. The resulting slurry was filtered and the wet cake was washed with 2 • propanol's dried in a vacuum oven at 50 ° C under N 2 purge overnight until a constant weight was reached. Separation of a tan solid (5 % yield, 97.5 % AUC). Other impurities are the bromine regioisomer (14% auc) and the second 155839.doc •70·201204720 bromine adduct (1.1% AUC). NMR (500 MHz, DMSO) δ 8.19 (1H, d, J = 8.1 Hz), 7.06 (br. s, 2 H), 6.64 (d, 1 H, 7 = 14.3 Hz). Example 3b: p-toluenesulfonium salt of (and)-1-((4-amino-2-bromo-5-fluorophenyl)amino)-3-(benzyloxy)propan-2-ol ) l^s^OBn

From

Catalytic amount Zn(C104)2-2H20 toluene, 80 ° C

2) H2, Pt(S)/C IPAc

3) TsOH-H20 DCM The following contents were fed into a flask sufficiently dried under A: activated powdered 4 A molecular sieve (50 wt% based on 2-bromo-5-fluoro-4-nitroaniline), 2- Bromo-5-fluoro-4-nitroaniline (1.0 eq.), zinc perchlorate dihydrate (2 〇 mol%) and toluene (8 vol). Mix the mixture for up to 3 minutes at room temperature. Finally, (R)-benzylglycidyl ether (2 Torr) in toluene (2 volumes) was added as a stabilizer. The reaction was heated to 80. (: (internal temperature) and stirred for about 7 hours or until 2-bromo-5-fluoro-4-nitromoutamine was &lt; 5% AUC. The reaction was cooled to room temperature and Celite 8 (5 〇 wt% was added) Ethyl acetate (10 vol.) was then added. The resulting mixture was filtered to remove (8) and molecular sieves and washed with ethyl acetate (2 vol.). /〇w/v) Washing filtrate. The organic layer was washed with a sodium bicarbonate solution (4 volumes χ25 〇/.w/v). The organic layer was concentrated on a rotary evaporator in vacuo. Isopropyl ester (1 〇 volume), and this solution was transferred to a Brookfield hydrogenator. 155839.doc •71· 201204720 Feeding 5 wt% Pt(S)/C (1.5 mol%) into the hydrogenator, And the mixture was stirred under N 2 at 30 C (internal temperature). The reactant was blown with n2, followed by boiling with hydrogen. The hydrogenator pressure was adjusted to 1 bar of hydrogen and the mixture was quickly (&gt; 12 rpm). At the end, the catalyst was filtered through a pad of, for example, 2, and washed with di-methane (10 vol). The filtrate was concentrated in vacuo. 2 volumes) trace any residual isopropyl acetate and concentrate to dryness on a rotary evaporator. The resulting residue was dissolved in dioxane (1 volume). Add p-toluenesulfonic acid monohydrate (1.2 equivalents) and Stir overnight. Filter the product and wash with di-methane (2 vol) and suction dry. Transfer the wet cake to a dry pan and place in a vacuum oven and dry under &amp; The constant weight was reached. The amine group 2-bromo-fluorophenyl)amino)-3-(n-decyloxy)propan-2-ol p-toluate was isolated as an off-white solid. Example 3c: (3-a-3-methylbutynyl)trimethyldecane

Propargyl alcohol (1.0 equivalents) was fed into the vessel. Aqueous hydrochloric acid (37%, 3.75 vol) was added and stirring was started. During the dissolution of the solid alcohol, a slight endotherm (5C to 6. 〇. The resulting mixture was stirred overnight for 6 hours) was observed, which slowly turned dark red. Water (5 vol) was fed into the 30 L jacketed vessel, which was then colded to i〇t. The internal temperature of the mixture was maintained below 25. The reaction mixture was slowly transferred to water under vacuum. The calcined (3 vol) was added and the resulting mixture was allowed to stand for 0.5 hr. The phases were deposited and the aqueous phase (10) value &lt;1) was drained and discarded. The organic phase was concentrated in a vacuum using a rotary evaporator to give the product as a red oil (yield: I55839.doc • 72· 201204720). Example 3d: (4-(Benzyloxy)_3,3-dimethylbutynyl)trimethyldecane

乂 1· Mg ►

TMS 2. BnOCH2Cl TMS

Method A All equivalent and volume descriptors in this section are based on 25 〇 g of reactant. Magnesium turnings (69.5 g, 2.86 mol, 2.0 equivalents) were fed into a 3 L·4 neck reactor and stirred with a magnetic stirrer under nitrogen for 5 hours. The reactor was immersed in an ice water bath. A solution of propargyl gas (25 〇g, 丨43 〇1,1·〇 equivalent) in THF (1.8 L, 7.2 vol) was slowly added to the reaction thief under stirring until the initial exotherm was observed ( The solution was determined by lpc: using ^-NMR spectroscopy to determine the formation of the Grignard reagent. After the exotherm was reduced, the remaining solution was slowly added while maintaining the batch temperature &lt; 15 C. The addition took about 35 hours. The resulting dark green mixture was decanted into a 2 l bottle. All equivalents and volume descriptors in this section were based on 5 〇〇g of reactant. Benzyl methyl methyl ether was fed to the 22 L reactor ( A solution of 95%, 375 g, 2.31 m〇1, 0.8 eq. in THF 〇5 L, 3 vol). The reactor was cooled in an ice X' valley. Two batches of the Grignard reagent batch prepared as above were combined and then, after maintaining the batch temperature below 25 〇c, via the addition funnel, the solution was added to the awkward base gas. Adding takes 1 · 5 hours. The reaction mixture was allowed to be stirred overnight (1 6 hours). All equivalent and volume descriptors in this section are based on the i h reactant. A 15% ammonium hydride solution (1 $ 0 in 8 $ ^ water 155839.doc -73· 201204720, ι volume) was prepared in a 3 〇 L jacketed reactor. The solution was cooled to 5 °C. Two parts of the Grenner reaction mixture prepared as above were combined and then transferred to a vaporized ammonium solution ten via a header vessel (hea (jei&gt; vessel), an exotherm was observed in this quenching, which was maintained to maintain the internal temperature The rate was lower than 25 ° C. After the transfer was completed, the temperature of the trough jacket was set to 25 ° C. Hexane (8 [, 8 volumes] was added and the mixture was stirred for 0.5 hours. After the phases were deposited, the water was discharged. Phase (pH 9) and discard. Wash the remaining organic phase with water (2 L '2 volumes). Use 22 l in vacuum

The organic phase was concentrated on a rotary evaporator to give a crude product as an orange oil. Method B Magnesium turnings (106 g, 4.35 mol, 1. 〇 equivalent) were fed into a 22 L reactor and then suspended in THF (760 mL, 1 vol). The vessel was cooled in an ice water bath to bring the batch temperature to 2 t. A solution of propargyl chloride (760 g, 4.35 m〇1, in stock) in THF (4.5 L, ό volume) was slowly added to the reactor. After the addition of 100 mL, the addition was stopped and the mixture was stirred until 13 C exotherm was observed, indicating the production of the Grenner reagent. After the exotherm subsided, a 500 mL propargyl gas solution was slowly added while maintaining the batch temperature &lt; 2 (TC). The Grenner reagent formation was determined by IPC using 1 H-NMR spectroscopy. Maintaining the batch temperature &lt; 2 (At TC, the remaining propargyl gas solution was slowly added. The addition took about 1.5 hours. The dark green solution obtained was mixed for 0.5 hours. The formation of the Grignard reagent was determined by iCP using 1H-NMR spectroscopy. Pure benzyl chloromethyl group The ether was fed into the reactor addition funnel and then added dropwise to the reactor while maintaining the batch temperature below 25° C. The addition took 1 hour. The reaction mixture was stirred overnight. The same procedure as in Method A was used. The relative amounts of the materials are subjected to aqueous treatment and concentration to give the product as an orange oil. 155839.doc •74· 201204720 Example 3e: 4-Benzyloxy-3,3-dimethylbutane•Alkyne

TMS

KOH MeOH --- 88%, decyl alcohol (6 vol) was fed to a 3 〇 L jacketed reactor via two steps of OBn, which was then cooled to 5 °C. Potassium hydroxide (85%, 丨. 3 equivalents) was added to the reactor. When the potassium hydroxide was dissolved, an amount of heat was observed to (9). Set the jacket temperature to 25 °C. Add a solution of 4-benzophenoxy_3,3-dimethyl-indole-trimethylsulfonyl-butane--alkyne (1·〇 equivalent) in methanol (2 vol) and stir the resulting mixture until HPLC The monitored reaction was completed. The typical reaction time at 25 is 3 to 4 hours. The reaction mixture was diluted with water (8 vol) and then stirred for 5 hours. Hexane (6 vol) was added and the resulting mixture was stirred for 5 hours. The phases were deposited and the aqueous phase was then drained (pH 1 (M1) and discarded. The organic phase was washed successively with a solution of KOH (85%, 〇. 4 eq.) in water (8 vol), water (8 vol). The organic phase is then concentrated using a rotary evaporator to give the title material as a yellow-yellow oil. The typical purity of this material is in the range of 8%, with a single impurity present. iH NMR (4 〇〇 MHz, CsDd δ 7.28 ( d, 2 Η, J=7.4 Hz), 7.18 (t, 2 H, J=7.2 Hz), 7.10 (d, 1H, J=7.2 Hz), 4.35 (s, 2 H), 3.24 (s, 2 H ), 1.91 (s, 1 H), 1.25 (s, 6 H). Example 3f . (R)_1-(4.Amino-2-(4-(benzyloxy)-3,3-dimethyl Butyl-1-ynyl)-5-fluoroanilino)_3_(benzyloxy)propan-2-ol 155839.doc •75· 201204720

(R) 1-(4-Amino-2-bromo-5-fluoroanilinyl) by stirring in dioxane (5 vol) and saturated NaHC03 solution (5 vol) until a clear organic layer is obtained The tosylate salt of -3-(benzyloxy)propan-2-ol is converted to the free base. The resulting layer was separated and washed with EtOAc EtOAc (EtOAc) ) 3-(Benzyloxy)propan-2-ol (free base). Palladium acetate (0.01 eq.), dppb (0.015 eq.), Cul (0.015 eq.) and potassium carbonate (3 eq.) were suspended in acetonitrile (1.2 vol). After stirring for 15 minutes, a solution of 4-benzyloxy-3,3-dimethylbut-1-yne (1.1 equivalent) in acetonitrile (0.2 vol) was added. The mixture was sparged with nitrogen for 1 hour, and then (/^-1-((4-amino-2-bromo-5-fluorophenyl)amino)-3-(phenylmethoxy)propane-2- A solution of the alcohol free base (1 eq.) in acetonitrile (41 vol.). The mixture was sparged with nitrogen for 1 hour and then heated to 8 (rc) monitored by HPlC and the reaction was usually completed in 3 to 5 hours. The mixture was cooled to room temperature and then filtered over celite. The filter cake was washed with acetonitrile (4 vols). The combined filtrate was azeotroped to dryness and then the mixture was then finely filtered to the next. Obtained amino group 2_(4_(phenylhydroxy)_3,3-dimethylbutan-1-ynyl-1-yl)-5-fluorophenyl)amino)_3 (benzyloxy)propane_2 The acetonitrile solution was used directly in the next procedure (cyclization) without further purification. 155839.doc •76- 201204720 Example 3g: (R)_i_(5-Amino-2·(1_(benzyloxy)_2_methylpropan-2-yl)-6-fluoro-1H-indole-1 -yl)-3-(benzyloxy)propan-2-ol

(MeCN)2PdCI2 Cul

Bis-acetonitrile dichloropalladium (0.1 eq.) and CuRO.i equivalent) were fed into the reactor' and then suspended in the above-obtained amino-2-(4-(benzyloxy)-3,3- a solution of dimethyl butyl _ _ _ _ _ _ _ _ _ _ _ _ _ _ fluorophenyl) amide) _ 3 _ (benzyloxy) propan-2-ol (1 equivalent) in acetonitrile (total of 9 5 volumes) . The mixture was sparged with nitrogen for 1 hour and then heated to 8 (rc. The progress of the reaction was monitored by Hplc and the reaction was usually completed in 1 to 3 hours. The mixture was filtered over celite and the cake was washed with acetonitrile. Ethyl acetate (75 vol.). The ethyl acetate solution was washed successively with &gt;^3_&gt;1114 (:1 aqueous solution (2 &gt;&lt; 2.5 vol), 1 〇〇 /0 brine (2.5 vol). The ester solution was stirred for 6 hours with 矽 〇 (8 wt equivalent) and Si-TMT (0.1 wt equivalent). After filtration, the resulting solution was concentrated. The residual oil was dissolved in DCM / heptane (4 vol) and then borrowed Purification by column chromatography. The oil thus obtained was crystallized from 25% EtOAc / heptane (4 vol). Crystals (8) small (5. Aminos. 2-Methylpropan-2-yl)-6-fluoro-1H-0-exo-i_yl)_3·(phenoxy)propan-2-ol is usually obtained in a yield of 27% to 38%. (400 MHz, DMSO) 7.38-7.34 (m, 4 Η), 7.32-7.23 (m, 6 Η), 7.21 (d, 1 H, /=12.8 Hz), 6.77 (d, 1H, J=9.0 Hz) s 6.06 (s, 1 H), 5.13 (d5 1H9 /=4.9 Hz), 4.54 (s5 2 H), 4.46 (br. s , 2 H), 4.45 155839.doc •77· 201204720 (s5 2 Η), 4.33 (d, 1 Η, 7=12.4 Hz), 4.09-4.04 (m, 2 H), 3.63 (d, 1H, J= 9.2 Hz), 3.56 (d, 1H, /=9.2 Hz), 3.49 (dd, 1H, 9.8, 4.4 Hz), 3.43 (dd, 1H, /=9.8, 5.7 Hz), 1.40 (s, 6 H). IIX.4.b. Coupling Example 3h: Synthesis of WN-(1_(3_(benzyloxy)_2hydroxypropyl(benzyloxy)-2-methylpropan-2-yl)·6-gas-1 Good "inducing _5_ base" small (22_difluorobenzo[rf][l,3]dioxol _5·yl)cyclopropanol

Soci2 W

1-(2,2-Difluoro-1,3-benzodioxol-5-yl)-cyclol (1.3 eq.) in toluene (2.5 vol., ^(2,2-difluoro^) A slurry was prepared in 3 benzodioxol-5-yl)-cyclopropanecarboxylic acid. Thionite gas (SOCh '1.7 equivalents) was added via an addition funnel and the mixture was heated to 6 (rc. The resulting mixture was stirred for 2 hours with propanecarboxylic acid. Toluene and excess SOC12 were distilled off using a rotary evaporator. Toluene was added (2.5 volumes). , based on 1_(2,2-difluoro-1,3-benzodioxole-5-yl)-cyclopropanecarboxylic acid) and distilling the mixture to 1 volume of toluene. -(5-Amino-2-(1-(benzyloxy)·2·decylpropan-2-yl)-6-fluoro-1Η-°ΒΒ-1·yl)-3-(benzene A solution of decyloxy)propan-2-ol (1 eq.) and triethylamine (3 eq.) in DCM (4 vol) was cooled to hydrazine. EtOAc was added to the acid toluene solution (1 vol) while maintaining The batch temperature was below 1 〇 ° C. The progress of the reaction was monitored by HPLC and the reaction was usually completed in a few minutes. After heating I55839.doc •78- 201204720 to 25 C, 5% NaHC03 (3.5 vol), 1 μ The reaction mixture was washed with NaOH (3.5 vol) and 1 M HCl (5 vol). The solvent was exchanged to methanol (2 vol) and (i 〇-N-(l-(3•(benzophenoxy)_2·hydroxypropyl) )_2_(1•(phenylhydroxy)-2-indenyl _2-yl)_6-fluoro_丨开-吲哚_5_基卜^^^Difluorobenzo[c?][l,3]dioxol-5-yl)cyclopropanoguanamine The resulting solution in decyl alcohol was used in the next step (hydrogenolysis) without further purification. Example 3i: Synthesis of compound 3

5% palladium/charcoal (about 5% wet, 1 equivalent of hydrazine) is fed to a suitable hydrogenation vessel. Carefully add (/?)_N_(1_(3_(benzyloxy)_2-hydroxypropyl)-2-(1-(phenylhydroxy)_2-methylpropan-2-yl) obtained above -, fluoro^仏吲哚_5_yl)-1-(2,2-difluorobenzodioxol-5-yl)cyclopropanecarbamide in methanol (2 vol), followed by 3 A solution of M HC in methanol. The vessel was purged with nitrogen and then purged with hydrogen. The mixture was stirred vigorously until completion of the reaction as determined by HPLC analysis. A typical reaction time is 3 to 5 hours, and the reaction mixture is passed through a solution of methanol and washed with methanol (2 volumes). The solvent was exchanged to isopropanol (3 volumes of crude compound 3 from 75% IPA•heptane (4 volumes, ie 1 volume of heptane added to 3 volumes of IPA) crystallized and the resulting crystals were at 50% IPA_ The heptane (i.e., 2 volumes of heptane is added to the mixture) is matured. The typical yield of compound 3 obtained by a two-step deuteration/hydrogenolysis procedure is in the range of 68% to 84%. The same procedure is used to recrystallize compound 3 from _^-heptane. 155839.doc 79·201204720 Compound 3 is also available from US Published Application No. US 2009/0131492, which is incorporated herein by reference. One of several synthetic routes disclosed in the preparation. Table 3-1: Physical data of Compound 3

521.5 1.69 1H NMR (400.0 MHz, CD3CN) d 7.69 (d, J=7.7 Hz, 1H), 7.44 (d, J=1.6 Hz, 1H), 7.39 (dd, J=1.7, 8.3 Hz, 1H), 7.31 (s, 1H), 7.27 (d, J=8.3 Hz, 1H), 7.20 (d, J=12.0 Hz, 1H), 6.34 (s, 1H), 4.32 (d, J=6.8 Hz, 2H), 4.15 -4.09 (m, 1H), 3.89 (dd, J=6.0, 11.5 Hz, 1H), 3.63-3.52 (m, 3H), 3.42 (d, 3=4.6 Hz, 1H), 3.21 (dd, J=6.2 , 7.2 Hz, 1H), 3.04 (t, J=5.8 Hz, 1H), 1.59 (dd, J=3.8, 6.8 Hz, 2H), 1.44 (s5 3H), 1.33 (s,3H)&amp;1.18 (dd , J=3.7, 6.8 Hz, 2H) ppm. _ III. Formulations In one aspect, the invention provides a formulation comprising a component selected from any of the embodiments described in column A of Table I and any one selected from column B of Table I The components of the examples and/or components selected from any of the examples described in column C of Table I. For your convenience, please show Table I here»

Table IA, column B, block C, example 1. 1 Γ 眚 眚 段落 段落 段落 段落 段落 段落 段落 段落 II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II Compound II.A.2. Compound 1 II.B.2. Compound 2 II.C.2. Compound 3 In one embodiment of this aspect, the formulation comprises one of the ones described in column A 155839.doc 80 · 201204720 Example and one of the embodiments described in column B. In another embodiment, the formulation comprises one of the embodiments described in column A and one of the embodiments described in C. In another embodiment, the formulation comprises a combination of one of the embodiments described in column A, one of the embodiments described in column B, and one of the embodiments of C. 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. In one embodiment of this aspect, the component of column B is a hydrazine compound. In another embodiment, the B barrier component is Compound 2. In one embodiment of this aspect, the component of column C is a hydrazine compound. In another embodiment, the component of column C is compound 3. In one embodiment, the formulation comprises a homogeneous mixture comprising the composition of the Table. In another embodiment, the formulation comprises a non-inulin mixture comprising a top composition. The pharmaceutical compositions of Table I can be administered or administered separately in a vehicle A. In some embodiments, the pharmaceutical compositions optionally comprise a pharmaceutically acceptable carrier, adjuvant or vehicle. In certain embodiments, such compositions further comprise one or more additional therapeutic agents, as appropriate. It will also be appreciated that certain compounds of the invention may be used in the free form of the treatment or, where appropriate, in the form of their pharmaceutically acceptable derivatives or prodrugs. A pharmaceutically acceptable derivative or prodrug according to the invention includes, but is not limited to, a pharmaceutically acceptable salt, vinegar, a salt of such vinegar, or after administration to a patient in need thereof (iv) directly or Any other adduct or derivative 155839.doc •81 · 201204720 which indirectly provides an alpha species or a metabolite or residue thereof as otherwise described herein. The term "pharmaceutically acceptable salt" as used herein means in the context of sound medical judgment, suitable for use in contact with humans and tissues of lower animals without excessive toxicity, irritation, allergic reactions and the like, and Salt commensurate with reasonable benefit/risk ratio. "Pharmaceutically acceptable salt" means any non-toxic salt of a compound of the present invention or a salt thereof, which, after administration to a recipient, is sufficient or indirectly to provide a compound of the present invention or an inhibitory active metabolite thereof or The residue. Pharmaceutically acceptable salts are well known in the art. For example, a ' SM Berge et al., in 乂Sciences, 1977 &lt; 5 (5, 1 -19, describe pharmaceutically acceptable salts in detail, which is incorporated herein by reference. Acceptable salts include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are those formed from amine groups with inorganic or organic acids, such as inorganic acids such as Hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and peroxyacids, such as acetic acid, oxalic acid, cis-succinic acid, tartaric acid, citric acid, succinic acid or malonic acid; or by using the technology Salts formed by other methods such as ion exchange used. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, besylate, benzoate, Hydrogen sulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentane propionate, digluconate, lauryl sulfate, ethanesulfonate, Formate, fumarate, glucoheptanoate, glycine Phosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionic acid 155839.doc •82· 201204720 salt, lactate, laurel Acid salt, lauryl sulfate, malate, maleate, malonate, decane sulfonate, 2-naphthalene sulfonate, nicotinic acid salt, nitrate, oleate, grass Acid salt, palmitate, pamoate, pectate, peroxosulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearic acid a salt, a succinate, a sulfate, a tartrate, a thiocyanate, a p-toluenesulfonate, an undecanoate, a valerate, and the like. A salt derived from a suitable base includes an alkali metal salt, Alkaline earth metal salts, ammonium salts and N+CCi.4 alkylh salts. The invention also contemplates the quaternization of any basic nitrogen-containing groups of the compounds disclosed herein. This can be obtained by the four-level classification. Water-soluble or oil-soluble or dispersible products. Representative metal or alkaline earth metal salts include sodium salts, lithium salts, potassium salts, calcium salts, magnesium salts and the like. Acceptable salts include, where appropriate, non-toxic ammonium salts, quaternary ammonium salts, and the use of such as tooth ions, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates, and aryl groups. An amine cation formed by the relative ionicity of the sulfonate. As described above, the pharmaceutically acceptable composition of the present invention additionally comprises a pharmaceutically acceptable carrier, adjuvant or vehicle, as used herein, as used herein. Any and all solvents, diluents or other liquid vehicles, dispersing or suspending aids, surfactants, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants suitable for the desired (iv) dosage form and Its analogues.

Remington's Pharmaceutical, as described in, for example, 16th edition, e % MartiMMack PubIishing c〇., East 〇n, pa, i98_, for the formulation of various pharmaceutically acceptable compositions and for the preparation of such compositions Known technology. Unless any oral carrier medium, such as due to the production of 155839.doc • 83· 201204720, is medically acceptable

Acid, sorbic acid or potassium sorbate; any undesirable biological effect or otherwise subject to any other component of the composition is incompatible with the compound in a detrimental manner, otherwise it is used in a box 缃 #匕铝; Aluminum citrate; lecithin; serum: buffer substance, such as phosphate, glycine-saturated glyceride mixture of plant fatty acids; water; salt or electrolyte, such as protamine sulfate, disodium hydrogen phosphate, hydrogen-depleted hydrogen, gas Sodium, vinylpyrrolidone; poly-sodium, zinc salt, colloidal silica, sulphate, poly-polyacrylate, wax, polyethylene, polyoxypropylene-block polymer, lanolin, sugar , such as lactose, glucose and sucrose; starch, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth, malt, Gelatin; talc; excipients such as cocoa butter and suppositories; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol or polyethylene Ester, such as ethyl oleate and ethyl laurate; agar; buffer, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution ); ethanol and phosphate buffer solutions; and other non-toxic compatible lubricants, such as sodium lauryl sulfate and magnesium stearate; and colorants, mold release agents, coating agents, sweeteners, according to the judge's judgment Flavoring and flavoring agents, preservatives and antioxidants may also be present in the compositions. The pharmaceutically acceptable composition of the present invention can be administered to humans and other animals via 155839.doc -84 - 201204720 oral, rectal, parenteral, transcerebral, intravaginal, intraperitoneal, topical (eg by Powder, ointment or drops), buccal, orally or nasally, or in a similar manner, depending on the severity of the infection being treated. In certain embodiments, the compositions of the present invention may be administered from about 0. 01 mg to about 50 mg per kilogram of body weight per day, and preferably from about i to about 25 mg per kilogram of body weight per day, one or more times per day. Oral or parenteral administration to obtain the desired therapeutic effect. Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, mashes, and elixirs. In addition to the active compound of the composition, the liquid dosage form may contain inert diluents which are conventionally employed in the art, for example, water or other solvents, solubilizers and emulsifiers such as ethanol, isopropanol, ethyl carbonate, acetic acid Ethyl ester, cumene alcohol, benzoic acid benzoic acid, propylene glycol, guanidine-butanediol, dimethyl decylamine, oil (especially cottonseed oil, peanut oil, corn oil, germ oil, eucalyptus oil, castor oil and Sesame oil), glycerin, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents. Injectable preparations, such as sterile injectable aqueous or oily suspensions, may be employed in accordance with known techniques using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a non-toxic parenterally acceptable diluent or solvent, for example, a solution in hydrazine, 3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution (U.S.P.) and isotonic sodium chloride solution. In addition, sterile non-volatile 155839.doc •85- 201204720 oils are used as solvents or suspension media. For this purpose, any bland fixed oil may be employed including synthetic monoglycerides or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables. Injectable formulations can be, for example, by filtration through a bacteria-retained device or by incorporation of a bactericidal agent in the form of a sterile solid composition which is dissolved or dispersed in sterile water or other sterile injectable medium. Can be sterilized. To prolong the action of the compositions of the present invention, it is often desirable to slow the absorption of compositions from subcutaneous or intramuscular injection. This can be achieved by using a liquid (4) float of a crystalline or amorphous substance having poor water solubility to achieve the 'absorption rate of the composition, depending on its rate of rotation, and its dissolution (d) may depend on crystal size and crystal form. . Alternatively, # delayed dissolution in the form of a composition for parenteral administration is achieved by dissolving or suspending the composition in an oil vehicle. Injectable reservoir forms are made by forming a microcapsule matrix of the composition in a biodegradable polymer such as polylactide-polyacetate. The release of the composition can be controlled depending on the ratio of the composition to the polymer and the nature of the particular polymer used (4). Other biodegradable polymerization (IV) examples include poly (original acid vinegar) and poly (acid field). A reservoir-injectable formulation is also preferably a test composition for rectal or vaginal administration by entrapment of the composition in liposomes or microemulsions compatible with body tissues, which may be by means of a netbook The compound of the invention is prepared by mixing with a suitable non-irritating excipient or (iv) such as cocoa butter, polyethylene glycol or a suppository, which is solid at ambient temperature but liquid at body temperature and thus in the vaginal canal Melting and releasing the active compound. A solid dosage form for oral administration includes capsules, troches, pills, powders and granules. In such solid dosage forms, the active compound is at least one such as a lemon. Inert, sodium or dicalcium phosphate inert, pharmaceutically acceptable excipients or carriers and/or mixtures of the following: a) fillers or extenders such as starch, lactose, cane, glucose, mannose Alcohol and citric acid; b) binders such as carboxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and gum arabic; C) humectants such as glycerin; d) disintegrants such as hydrazine Fat, carbonic acid #5, Potato or tapioca starch, alginic acid, certain salts and sodium carbonate; e) solution blockers such as paraffin; absorption enhancers such as quaternary ammonium compounds; g) wetting agents such as cetyl alcohol and single Glyceryl stearate; h) adsorbents such as kaolin and bentonite; and hydrazine) lubricants such as talc, calcium stearate, magnesium stearate, sodium, and mixtures thereof. In the capsule, a buffer may be included. In the case of solid polyethylene glycol, lauryl sulfate tablet and pellets, similar types of solid fines can also be used in the dosage form; A A _

J55839.doc •87· 201204720 Lactose and excipients of high molecular weight polyethylene glycol and its analogues. The active compound may also be in microencapsulated form with one or more excipients as described above. Solid dosage forms of lozenges, dragees, capsules, pills and granules can be prepared with coatings and shells, such as enteric coatings, release control coatings, and other coatings well known in the art. In such 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 contain, in addition to inert diluents, such as tableting lubricants and other tableting aids such as magnesium stearate and microcrystalline cellulose, as is conventional practice. In the case of capsules, lozenges and pills, the dosage form may also contain a buffer. It may optionally contain an opacifying agent, and may also be a composition which, in a delayed manner, only releases or preferentially releases the active ingredient in a certain portion of the intestinal tract. Examples of embedding compositions that can be used include polymeric enamels and waxes. Dosage forms for topical or transdermal administration of a compound of the invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches. The active component is admixed under sterile conditions with apharmaceutically acceptable carrier and any required preservative or buffer. Ophthalmic formulations, ear drops, and eye drops are also contemplated within the scope of the present invention. In addition, the present invention encompasses the use of transdermal patches for the additional advantage of delivering compounds to the body. These dosage forms are prepared by dissolving or dispensing the compound in a suitable medium. 4 An absorption enhancer can be used to increase the transdermal flux of the compound. The rate can be controlled by providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel. σ, other needs should also be understood, the composition disclosed herein can be combined with one or more 155839.doc -88 - 201204720 or medical procedures, before or after the cast (4)) It should be understood that the phase of the grain used and the desired therapeutic effect to be achieved. Also, the therapy can achieve the desired effect on the same condition (e.g., the compound of the invention can be administered simultaneously with another agent used to treat the same condition) or eight (e.g., to control any adverse effects). As used herein, other therapeutic agents that are normally administered to treat or prevent a particular disease or condition are referred to as "the money or condition to be treated", and in the examples, other agents are selected from the group consisting of mucolytic agents, Bronchodilators, antibiotics, anti-infectives, anti-inflammatory agents, CFTR modulators or nutrients other than the compounds of the invention. In one embodiment, 'other agents are antibiotics. Exemplary antibiotics for use herein include tobramycin (t 〇bramyCin), including tobramycin inhalation type sputum (TIP), azithromycin; aztreonam 'atomized form of aztreonam; amikacin' including its lipid Phytofloxacin, including its formulation for administration by inhalation; levofloxacin, including its nebulized formulation, and two antibiotics (eg, fosfomycin) A combination of tobramycin. In another embodiment, the other agent is a mucolytic agent. Exemplary mucolytic agents suitable for use herein include Pulmozyme®. In another embodiment, other The agent is a bronchodilator. Exemplary bronchodilators include aibutero1, metaproterolol sulfate, acetic acid, pirbuterol (155839.doc -89-201204720 acetate), salmeterol ( Salmeterol) or tetrabuiine sulfate 〇 In another embodiment, other agents are effective in restoring the fluid on the surface of the lung trachea. These agents improve the movement of salt into and out of the cell, resulting in a higher hydration of the mucus in the lung trachea. And thus easier to remove. These exemplary agents include high ginseng physiological saline, denufosol tetrasodium ([[(3S,5R)-5-(4-amino-2- side oxygen) Alkylpyrimidine-i-yl)_3_-yloxyoxol-2-yl]methoxy-based thiol][[[(2R,3S,4R,5R)-5-(2,4- Bis-oxypyrimidin-1-yl)_3,4-dihydroxyoxol-2-yl]methoxy-hydroxyphosphonyl]oxy-hydroxyphosphonium]hydrogen phosphate), or bronchol (bronchitol) (inhalation formulation of mannitol). In another embodiment, the other agent is an anti-inflammatory agent, and the agent for reducing inflammation in the lungs is suitable. Such exemplary agents include ibuprofen, docosahexaenoic acid (DHA), sildenafil, inhaled glutathione, pioglitazone (pi〇gHtaz〇ne), hydroxy Hydroxychloroquine or Simavastatin. In another embodiment, the other agent is a CFTR modulator other than Compound 1, i.e., an agent having an effect of modulating CFTR activity. Such exemplary agents include ataluren ("PTC124®"; 3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic acid), West Sinapultide, lancovutide, depelestat (human recombinant neutrophil elastase inhibitor), test D bis (C〇bipr〇St〇ne) (7-{(2R,4aR,5R,7aR)-2-[(3S)-l,l-difluoro-3·methylpentyl]-2·hydroxy-6-oxooxy octahydrocyclopentane Alkeno[b]pyranyl-5-yl}g 155839.doc -90· 201204720 acid) or 3-(6-(1-(2,2·diIbenzo[dni,3]dioxole) Dilute _5_yl) cyclopropanthocarbylamino)-3-methyl-_2-yl)benzoic acid. In another embodiment, the other agent is 3-(6-(1-(2,2-di-benzobenzo[d][1,3]dioxol-5-yl)cyclopropane oxime Amino)_3_methylpyridine-2-yl)benzoic acid. In another embodiment, the other agent is a nutrient. Such exemplary agents include pancreatic lipase (pancreatic enzyme substitute) including pancreas8, Pancreacarb®, Ultrase® 4Cre〇n8; Upr〇t〇mase8 (previously Trizytek®), Aquadeks® or glutathione inhalation Agent. In one embodiment, the other nutrient is pancreatic lipase. The amount of other therapeutic agent present in the compositions of the present invention should be no greater than that normally employed in compositions comprising the therapeutic agent as the sole active agent. Preferably, the amount of the therapeutic agent in the compositions disclosed herein is from about 50% to about 1% of the amount normally present in the composition comprising the agent as the sole therapeutically active agent. Within the scope. Compositions of the invention as disclosed herein may also be incorporated into compositions for coating implantable medical devices such as prosthetic, prosthetic valves, vascular grafts, vascular stents, and luminaires. Accordingly, in another aspect, the invention includes a composition for coating an implantable device comprising a composition as disclosed herein or a pharmaceutically acceptable composition thereof, and classes and subclasses herein The latter, and a carrier suitable for coating the implantable device. In another aspect, the invention includes an implantable device coated with a composition comprising a composition as described herein, or a pharmaceutically acceptable composition thereof, and a carrier suitable for coating the implantable device Compositions. Suitable preparation methods for coating agents and coated implantable devices are described in U.S. Patent Nos. 6, 〇99,562; 5,886, 〇26; and 155839.doc •91 · 201204720 5,304,121. The coating agent is usually a biocompatible polymer gel polymer, polymethyldioxane, &amp; water/lipid polycaprolactone, polyethylene glycol, polylactic acid, ethylene vinyl acetate and mixture. The coating may optionally be covered by a suitable outer coating of fluoropolysulphate, polyfluorene, polyethylene glycol, dish grease or combinations thereof to impart controlled release characteristics to the composition. In order to more fully understand the invention described herein, the following examples are set forth. It is to be understood that the examples are for illustrative purposes only and are not intended to limit the invention in any way. IV. Method of Use In another aspect, the invention provides a method of treating a condition, disease or condition involving &amp; CFTR comprising a compound of formula 1 and a compound of formula π and/or a compound of formula III. The method comprises administering Formulations for individuals (preferably sprayed animals) in need. In the examples, the pharmaceutical composition comprises Compound a Compound 2. In another embodiment, the pharmaceutical composition comprises Compound i and Compound 3. In another embodiment, the pharmaceutical composition comprises Compound Compound 2 and Compound 3. In another embodiment, the pharmaceutical composition comprises the components as provided in the specification. In certain embodiments, the invention provides a method of treating a condition, disease, or condition involving a deficiency in the activity of 〇^111. The method comprises administering to a subject, preferably a mammal, a pharmaceutical composition of the invention. In another aspect, the invention provides methods of treating or lessening the severity of a condition, disease or condition involving a CFTR mutation. In certain embodiments, the invention provides a method of treating (a method of a condition, disease or condition characterized by a deficiency in paw activity) comprising administering to a subject in need thereof, preferably a mammal, a pharmaceutical composition of the invention. .doc -92. 201204720 In another aspect, the invention also provides a method of treating or lessening the severity of a disease in a subject, the method comprising administering to a subject in need thereof, preferably a mammal, a pharmaceutical composition of the invention And the disease is selected from cystic fibrosis, asthma, smoking-induced COPD, chronic bronchitis, sinusitis, constipation, pancreatitis, pancreatic insufficiency, caused by congenital bilateral vas deferens (CBAVD) Male infertility, mild lung disease, idiopathic pancreatitis, allergic bronchi and pulmonary aspergillosis (ΑΒΡΑ), liver disease, hereditary emphysema, hereditary hemochromatosis; coagulation-fibrinolysis deficiency, such as c protein Defect; type 1 hereditary angioedema; lipid treatment defects, such as familial hypercholesterolemia; type 1 chylomicronemia, no beta lipoproteinemia; lysosomal storage disease, such as I Cellular disease/pseudo-Hurler; mucop〇lysaccharid〇ses, Sandhof/Tay_Sachs, π-gram Crigler-Najjar, polyendocrine/hyperinsulinemia, diabetes, Laron dwarfism, myeloperoxidase deficiency, primary parasympathetic hypofunction, melanin Tumor, is glycolytic CDG (glycan〇sis CDG type 1), congenital dysfunction of the dysfunction, osteogenesis imperfecta, hereditary hypocytoplasmic jk, ACT deficiency, diabetes insipidus (DI), neurophysiology DKneurophyseal DI), kidney-derived DI, Charcot-Made Tooth syndrome, perHzaeus·Merzbacher disease; neurodegenerative diseases such as Alzheimer's disease (Alzheimer's disease), Parkinson's disease disease, amyotrophic lateral sclerosis, progressive nuclear anesthesia, Pick's disease; several poly-mytamine neurological disorders, such as Hending 155839.doc •93- 201204720 Huntington's disease, type I spinal cerebellum Dysregulation, spinal medullary muscle atrophy, dentate nucleus, dentatorubal pallidoluysian and muscular dystrophy, and spongiform encephalopathy, such as Creutzfeldt_Jak〇b disease ( Due to defects in prion protein treatment), Fabry disease, Straussler_Scheinker syndrome, COPD, dry eye disease or Sjogren's disease (Sj〇gren, s disease), osteoporosis, osteopenia, bone healing and bone growth (including bone repair, bone regeneration, reduction of bone resorption and increased bone deposition), Gorham's Syndrome; chloride channel disease, such as innate Myotonic stiffness (Thomson and Becker forms), Type III Bartter's syndrome, Dent's disease, epilepsy, hyperekplexia, lysosomal storage Accumulation disease, Angelman syndrome and primary ciliary dyskinesia (PCD) (a structure about cilia and / Or a functional hereditary disorder term, including PCD (also known as Kartagener syndrome) with visceral retrograde, PCD without visceral retrograde, and cilia hypoplasia. In some embodiments, the method comprises treating or lessening the severity of cystic fibrosis in a patient&apos; comprising administering to the patient a composition as defined herein. In certain embodiments, the patient has a mutant form of human CFTR. In other embodiments, the patient has one or more of the following mutations ΔF508, R117H, and G5 51D of human CFTR. In one embodiment, the method comprises treating a cyst of a patient having an AF508 mutation of human CFTR 155839.doc 94· 201204720 Sexual fibrosis or reducing the severity thereof, comprising administering to the patient a composition as defined herein. In one embodiment, the method comprises treating or reducing the severity of cystic fibrosis in a patient having a G5 51D mutation of human CFTR comprising administering to the patient a composition as defined herein. In a consistent embodiment, the method comprises treating or lessening the severity of cystic fibrosis in a patient having an AF508 mutation of human CFTR on at least one dual gene comprising administering to the patient a composition as defined herein. In one embodiment, the method comprises treating or lessening the severity of cystic fibrosis in a patient having a ΔΡ508 mutation of human CFTR on two dual genes comprising administering a composition as defined herein to the patient. In one embodiment, the method comprises treating or lessening the severity of cystic fibrosis in a patient having a G551D mutation of human CFTR on at least one dual gene comprising administering a composition as defined herein to the patient. In one embodiment, the method comprises treating or reducing the severity of cystic fibrosis in a patient having a G55 1D mutation of human CFTR on two dual genes comprising administering a composition as defined herein to the patient . In some embodiments, the method comprises reducing the severity of cystic fibrosis in the patient comprising administering to the patient a composition as defined herein. In certain embodiments the patient has a mutated form of human CFTR. In other embodiments, the patient has one or more of the following mutations ΔΙ?5〇8, R117Η, and G551D of human CFTR. In one embodiment, the method comprises reducing the severity of cystic fibrosis in a patient having an AF508 mutation in human CFTR comprising administering to the patient a composition as defined herein. In a consistent example, the '§海 method includes reducing the severity of cystic fibrosis in a patient having a human CFTR 155839.doc • 95-201204720 G55 1D mutation, which comprises administering to the patient a composition as defined herein . In one embodiment, the method comprises reducing the severity of cystic fibrosis in a patient with AF508 canine having human CFTR on at least one dual gene comprising administering to the patient a composition as defined herein. In one embodiment, the method comprises reducing the severity of cystic fibrosis in a patient having an AF508 mutation of human CFTR on two dual genes comprising administering to the patient a composition as defined herein. In one embodiment, the method comprises reducing the severity of cystic fibrosis in a patient having a G5 51D mutation of human CFTR on at least one dual gene comprising administering to the patient a composition as defined herein. In one embodiment, the method comprises reducing the severity of cystic fibrosis in a patient having a G55 1D mutation of human CFTR on two dual genes&apos; comprising administering to the patient a composition as defined herein. In some aspects the invention provides a method of treating or lessening the severity of osteoporosis in a patient comprising administering to the patient a composition as defined herein. In certain embodiments, a method of treating or lessening the severity of osteoporosis in a patient comprises administering to the patient a pharmaceutical composition as described herein. In some aspects, the invention provides a method of treating or lessening the severity of osteopenia in a patient comprising administering to the patient a composition as defined herein. In certain embodiments, a method of treating or reducing the severity of osteopenia in a patient comprises administering to the patient a pharmaceutical composition as described herein. 155839.doc • 96-201204720 In some aspects, the invention provides a method of causing bone healing and/or bone repair in a patient&apos; which comprises administering to the patient a composition as defined herein. In certain embodiments, a method of bone healing and/or bone repair in a patient comprises administering to the patient a pharmaceutical composition as described herein. In some aspects, the invention provides a method of reducing bone resorption in a patient&apos; which comprises administering to a patient a composition as defined herein. In some aspects, the invention provides a method of increasing bone deposition in a patient, which comprises administering to the patient a composition as defined herein. In certain embodiments, a method of increasing bone deposition in a patient comprises administering to the patient a composition as defined herein. In some aspects, the invention provides a method of treating or reducing the severity of a C〇pd in a patient comprising administering to the patient a composition as defined herein. In certain embodiments, treating the patient for COPD or reducing A method of its severity comprises administering to a patient a composition as defined herein. In some aspects, the invention provides a method of treating or lessening the severity of smoking-induced COPD in a patient comprising administering to the patient a composition as defined herein. In certain embodiments, a method of treating or lessening the severity of smoking-induced COPD in a patient comprises administering to the patient a composition as defined herein. In some aspects, the invention provides a method of treating or lessening the severity of chronic bronchitis in a patient comprising administering to the patient a composition as described herein. In certain embodiments, a method of treating or reducing the severity of chronic bronchitis in a patient comprises administering to the patient a composition as defined herein. According to an alternative embodiment, the invention provides a method of treating cystic fibrosis comprising the step of administering to the mammal a composition as defined herein. In accordance with the present invention, an "effective amount" of a composition is an amount effective to treat or ameliorate the severity of one or more of the diseases, disorders or conditions as described above. Another aspect of the invention provides a method of administering a pharmaceutical composition which is achieved by oral administration of a composition such as a composition as described herein at least once a day. In one embodiment, the method comprises administering to the patient a composition of Table I as defined herein once every 24 hours. In another embodiment, the method comprises administering to the patient a composition as defined herein once every 12 hours. In another embodiment, the method comprises administering to the patient three times a composition as defined herein. In another embodiment, the method comprises administering to the patient a composition as defined herein. In accordance with the methods of the present invention, the compositions can be administered using any amount and any route of administration effective to treat or ameliorate one or more of the diseases, disorders or conditions described above. In certain embodiments, the compositions of the present invention are useful for treating cystic fibrosis or reducing the severity of a patient exhibiting residual CFTR activity in the apical membrane of the respiratory and non-respiratory epithelium. The presence of residual CFTr activity at the epithelial surface can be readily detected using methods known in the art, such as standard electrophysiological techniques, biochemical techniques, or tissue chemistry techniques. These methods use in vivo or ex vivo electrophysiological techniques, assays for measuring the cell surface density of sweat or saliva or monitoring cell surface density by ex vivo biochemistry or histochemistry I55839.doc -98 - 201204720 techniques to identify CFTR activity. Using these methods, residual CFTR activity in a patient with a heterozygous or homotypic junction for a variety of different mutations, including patients with homozygous or heterozygous for the most common mutation AF508, can be readily described. In another embodiment, the compositions of the present invention are useful for treating or reducing the severity of cystic fibrosis in a patient having residual CFTR activity induced or enhanced by pharmacological methods or gene therapy. These methods increase the amount of CFTR present at the surface of the cell, thereby inducing the existing level of CFTR activity in the patient or increasing the level of residual CFTR activity in the patient. In one embodiment, a composition as defined herein may be adapted to treat certain genotypes that are indicative of residual CFTR activity (eg, scorpion mutations (regulated or gated impaired), class IV mutations (transduction changes), or Ν-type mutation (reduced synthesis)) (Lee R. Choo-Kang, Pamela L) Zeitlin, /,//, &quot;, IV, and V cystic fibrosis Transmembrane Conductance Regulator Defects and Opportunities of Therapy·, Current

Cystic fibrosis or reduced severity in patients within the scope of Opinion in Pulmonary Medicine 6:521-529, 2000). Other patient genotypes exhibiting residual Cftr activity include patients who are homozygous for one of these classes or heterozygous for any other class of mutations, including class I mutations, class 11 mutations, or unclassified mutations. In one aspect, the invention includes a method of treating a Class III mutation as described above, comprising administering to a patient in need thereof a composition comprising: a compound of Formula I and a compound of Formula II and/or a compound of Formula III One or both. In some embodiments of this aspect, the composition comprises a compound of formula I and a compound of formula 11 155839.doc-99-201204720. In some embodiments of this aspect, the composition comprises a compound of formula i and a compound of formula III. In some embodiments of this aspect, the composition comprises a compound of formula I and a compound of formula II and a compound of formula III. In another embodiment of this aspect, the pharmaceutical composition comprises Compound 1 and Compound 2. In another embodiment, the pharmaceutical composition comprises Compound 1 and Compound 3. In another embodiment, the pharmaceutical composition comprises Compound 1, Compound 2, and Compound 3. In one embodiment, a composition as defined herein may be adapted to treat a range of certain clinical phenotypes (eg, a moderate to mild clinical phenotype associated with the amount of residual CFTR activity in the epithelial apical membrane). Cystic fibrosis or severity of the patient within the patient. These phenotypes include patients who present with pancreatic insufficiency or who are diagnosed with idiopathic pancreatitis and congenital bilateral vas deferens or mild lung disease. The exact amount required will vary from individual to individual, depending on the species, age and condition of the individual, the severity of the infection, the particular agent, the mode of administration, and the like. Preferably, the compositions of the present invention are formulated in dosage unit form to facilitate dosage administration and homogeneity. The expression "agent 1 unit form" as used herein refers to a physical individual sputum suitable for the agent to be treated. It will be understood, however, that the total daily usage of the compositions of the present invention will be determined by the attending physician within reasonable medical judgment (4). The particular effective dosage for any particular patient or organism depends on a number of factors, including the severity of the condition and condition being treated; the activity of the composition employed; the age, weight, and general health of the particular group of patients employed. , sex and diet; = time, route of administration and (iv) rate of excretion of the composition; duration of treatment; combination with or in combination with the particular composition used, 155839.doc •100· 201204720 and in medical technology Similar factors are well known. The term "patient" as used herein means an animal, preferably a mammal, and most preferably a human. In one aspect, the invention provides a kit comprising a composition as defined herein. v. Detection method VA Scheme 1 Detection and measurement of AF508-CFTR enhancement characteristics of compounds. Detection of compound AF508-CFTR regulation characteristics of membrane potential optical method This detection method uses a fluorescent plate reader (for example, FLIPR III, Molecular Devices, Inc.) used fluorescent voltage sensing dyes to measure changes in membrane potential as a reading of increased functional AFSOS-CFTR in NIH 3T3 cells. The driving force for the reaction is the formation of a gas ion gradient, along with channel activation by a single liquid addition step after the cells have been previously treated with the compound and subsequently loaded with a voltage sensing dye. Identification of potentiator compounds To identify synergists of AFSOS-CFTR, a dual-addition HTS assay format was developed. This HTS assay uses a fluorescent voltage sensing dye to measure the change in membrane potential on FLIPR III as a measure of the increase in gate (conduction) of AF508 CFTR in AF508 CFTR NIH 3T3 cells. The driving force for the reaction is a cr ion gradient, followed by a single pass using a fluorescent plate reader (such as FLIPR III) after treatment with the cell previously synergist compound (or DMSO vehicle control) and subsequent loading of the redistributed dye. The channel caused by forskolin is activated in the liquid addition step. Solution 155839.doc -101 - 201204720 Electrolyte No. 1: (in mM) NaCl 160; KCl 4_5 ; CaCl 2 2 ; MgCl 2 1 ; HEPES 10, pH 7.4 (with NaOH). Chlorine-free electrolyte: The vaporized salt in the No. 1 electrolyte (described above) was changed to gluconate. Cell culture Optical measurement of membrane potential was performed using NIH3T3 mouse fibroblasts stably expressing ΔΡ508-ΟΡΤΚ_. In a 175 cm2 culture flask at 37 ° C in 5% CO 2 and 90% humidity, supplemented with 2 mM branic acid, 10% fetal bovine serum, sputum, β-ΜΕ, lx penicillin/streptomycin Cells were maintained in Dulbecco's modified Eagle's medium with 25 mM HEPES. For all optical assays, cells were seeded at approximately 20,000 cells per well in 384 wells coated with Matrigel and incubated at 37 °C for 2 hours, followed by 27. (: culture for 24 hours for synergist assay. For corrective assay, cells are cultured for 16 to 24 hours in the presence and absence of compounds at 27 ° C or 37 ° C. For detection of compounds △ Electrophysiological assay for F508-CFTR regulatory properties.

Vssing chamber assay A Ussing chamber experiment was performed on polarized tracheal epithelial cells expressing AF508-CFTR to further characterize the ΔΡ508-CFTR modulator identified in the optical assay. As described previously (Galietta, LJV, Lantero, S., Gazzolo, A., Sacco, 0·, Romano, L., Rossi, GA &amp; Zegarra-Moran, O. (1998) / « Ce//. Dev 5b/· 34, 478-481) Cultured bronchial tissue isolated non-CF and CF tracheal epithelium and inoculated onto Costar® SnapwellTM filter 155839.doc • 102· 201204720 pre-coated with NIH3T3 conditioned medium. After 4 days, the apical membrane medium was removed and the cells were allowed to grow at the air liquid interface for more than 14 days to be used. This produces a monolayer of fully differentiated columnar cells that have characteristic cilia (characterized by the tracheal epithelium). Non-CF HBE was isolated from non-smokers who did not have any known lung disease. CF-HBE was isolated from patients who were homozygous for ΔF508-CFTR. The HBE grown on the Costar® SnapwellTM cell culture insert was placed in a Using chamber (Physiologic Instruments, Inc., San Diego, Calif.) and a voltage clamp system (Department of Bioengineering, University of Iowa, IA) was used. Transepithelial electrical resistance and short circuit current (Isc) were measured in the presence of a gradient from the basolateral to the apical cr. Briefly, HBE was studied at 37 ° C under voltage clamp recording conditions (Vw = 0 mV). The basolateral solution contains (in mM) 145 NaCl, 0.83 K2HP〇4, 3.3 KH2P04, 1.2 MgCl2, 1.2 CaCl2, 10 glucose, 10 HEPES (pH adjusted to 7.35 with NaOH), and the top membrane solution contains ( In mM, 145 sodium gluconate, 1.2 MgCl2, 1.2 CaCl2, 10 glucose, 10 HEPES (pH adjusted to 7.35 with NaOH). Identification of Synergist Compounds A typical approach utilizes a basolateral to apical cr concentration gradient. To establish this gradient, a standard Ringer's solution was used on the basolateral membrane, and the apical membrane NaCl was changed to an equivalent molar concentration of sodium gluconate (titrated to pH 7.4 with NaOH) to obtain a wide range of Cl across the epithelium. Concentration gradient. Forskolin (10 μM) and all test compounds were added to the apical membrane side of the cell culture insert. The efficacy of the putative AFSOS-CFTR synergist was compared to the efficacy of the known synergist genistein. 155839.doc -103 - 201204720 Patch clamp recording is used as previously described (Rae, J" Cooper, K., Gates, P. and Wat sky, Μ· (1991) «/· TVeMroicz·· Mei/zods 37, 15-26) Perforated Diaphragm Recording Configuration Monitors total Cl· current in ΔΡ508-ΝΙΗ3Τ3 cells. Axopatch 200B Patch Sweet Amplifier is used at 22°C (Axon Instruments Inc.,

Foster City, CA) performs voltage clamp recording. The pipette solution contains (in mM) 150 iV-mercapto-D-glucosamine (NMDG)-Cl, 2 MgCl2, 2 CaCl2, 1 〇 EGTA, 10 HEPES and 240 pg/mL amphotericin _ B ( Amphotericin-B) (pH adjusted to 7.35 with HC1). The extracellular medium contained (in mM) 150 NMDG-CM, 2 MgCl2, 2 CaCl2, 1 〇 HEPES (pH adjusted to 7.35 with HCl). Pulse generation, data acquisition and analysis were performed using a PC equipped with a Digidata 1320 A/D interface and Clampex 8 (Axon Instruments Inc.). To activate AF508-CFTR, 10 μM of forskolin and 20 μL of genistein were added to the cell and the current-voltage relationship was monitored every 30 seconds. Identification of potentiator compounds The ability of AFSOS-CFTR potentiators to increase macroscopic ΔΡ508· CFTR Cr current (Uf5〇8) in NIH3T3 cells stably expressing AFSOS-CFTR was also investigated using a perforated patch recording technique. The synergistic effector identified by the optical assay enhances IAF 508 as the dose changes, and similar performance and efficacy are observed in optical assays. In all cells studied, the potential was reversed to about -30 mV before and during the application of the potentiator, which is the calculated Ecl (-28 mV). Cell Culture 155839.doc •104· 201204720 NIH3T3 mouse fibroblasts stably expressing AFSOS-CFTR were used in whole cell recording. In a 175 cm2 culture flask at 37 ° C in 5% CO 2 and 90% humidity, supplemented with 2 mM branic acid, 10% fetal bovine serum, sputum, β-ΜΕ, lx penicillin/streptavidin Cells were maintained in Duchenne's modified Ig's medium with 25 mM HEPES. For whole-cell recording, 2,500-5,000 cells were seeded on a glass cover slip coated with poly-L-lysine and incubated at 27 ° C for 24 to 48 hours before being used to test the activity of the synergist; Incubation at 37 ° C in the presence or absence of a corrective compound for measuring the activity of the corrective agent. Single channel recording uses the cut-out inner side outward as described previously. Diaphragm recording method (Dalemans, W., Barbry, P., Champigny, G., Jallat, S., Dott, K., Dreyer, D., Crystal, RG, Pavirani, A., Lecocq, JP., Lazdunski, M. (1991) iVaiwre 354, 526-528), using Axopatch 200B patch clamp amplifier (Axon Instruments Inc.) to observe the wt expressed in NIH3T3 cells - BRTR and temperature-corrected AFSOS-CFTR gated activity. The pipette contains (in mM): 150 NMDG, 150 days of aspartic acid, 5 CaCl2, 2 MgCl2 and 10 HEPES (pH adjusted to 7.35 with Tris base). The cell contains (in mM): 150 NMDG-CU, 2 MgCl2, 5 EGTA, 10 TES and 14 Tris base (pH adjusted to 7.35 with HC1). After excision, 1 mM Mg-ATP, 75 nM cAMP-dependent protein kinase catalytic subunit (PKA; Promega Corp. Madison, WI) and 10 mM NaF were added to inhibit protein phosphatase (which would prevent current reduction), Thus, the wt-CFTR and ΔF5 08-CFTR 活化 were activated to maintain the pipette potential at 80 mV. The channel activity was analyzed from a patch containing $2 live 155839.doc -105-201204720 sexual channels. The number of active channels during the pass process is determined by the maximum number of sync openings. To measure the single-channel current amplitude 'offline' at 100 Hz, filter from ΔΡ508 (: Ι7ΤΚ activity 12 sec.) and then use the data to use Bio-Patch Analysis software (Bi〇-L〇gic comp. France) Constructs a full-point amplitude histogram fitted with a multigaussian functi〇n. The total microscopic current and the open probability are measured from the channel activity of 2 〇2 (P. Use the biofilm software or the self-relationship Pc=I/i(N) (where 1 = average current, i = single channel current amplitude and N = number of active channels in the membrane) to determine P〇. NIH3T3 mouse fibroblasts with stable expression of △F508-CFTR were used for excision patch clamp recording. At 37 (in 5% C〇2 and 90% humidity in 175 cm2 culture flasks) Cells were maintained in Duchenne's modified Ig's medium with 2 mM facial acid, 1% fetal bovine serum, sputum, β-ΜΕ, lx penicillin/streptomycin, and 25 mM HEPES. For single channel recording, 2,500 to 5,000 cells were seeded onto a glass cover slip coated with poly-lysine and Incubation at 27 ° C for 24 to 48 hours. Example: Activity of the compound of formula I &lt; The compound of formula I is suitable as a modulator of the ATP-binding calo transporter. Examples of the activity and efficacy of the compound of formula J are shown in the table below. In the case of 1-2, if the activity is measured to be 2.0 μΜ or less, the activity of the compound is indicated by “+++”. If the activity is measured to be 2 μΜ to 5.0 μΜ, the activity of the compound is indicated by “++”, if active If the measurement is greater than 5 · 〇μΜ, the compound activity is indicated by "+", 155839.doc -106- 201204720 and if no data is available, the activity of the compound is indicated by "-". If the efficacy is calculated to be greater than 100%, then "+++" indicates the effect. If the effect is calculated to be 100% to 25%, the effect is indicated by "++". If the effect is calculated to be less than 25%, the effect is indicated by "+", and if no data is available, , the effect is indicated by "-". It should be noted that the 100 ° / ◦ effect is the maximum reaction obtained with 4-mercapto-2-(5-phenyl-1H-pyrazol-3-yl)phenol. 2 Activity and efficacy of the compound of formula I Example Activity % Efficacy Compound No. EC50 (nm) 1 -HH- -Η- 1-2 +-H- ++ 1-3 -Hl· -Η- 1-4 +++ ++ 1-5 +-H- -Η-+ 1-6 +++ 十++ 1-7 +++ -Η- 1-8 +++ -Η-1-9 + ++ 1-10 +++ +++ 1-11 +-H- ++ 1-12 +++ ++ 1-13 -HTMl· ++ 1-14 ++ VC scheme 2 detection and quantity A method for detecting the AF508-CFTR correcting property of a compound is a membrane potential optical method for detecting the AFSOS-CFTR regulating property of a compound. 155839.doc -107- 201204720 Optical membrane potential detection using Gonzalez and Tsien (see Gonzalez, JE and RY Tsien (1995) "Voltage sensing by fluorescence resonance energy transfer in single cells" Biophys J 69(4): 1272-80 And the voltage-sensitive FRET sensor described in Gonzalez, JE and RY Tsien (1997) "Improved indicators of cell membrane potential that use fluorescence resonance energy transfer" Chem Biol 4(4): 269-77), and Instruments for measuring fluorescence changes, such as voltage/ion probe readers (VIPR) (see Gonzalez, J. E., K. Oades et al., (1999) "Cell-based assays and instrumentation for screening ion-channel Targets" Drug Discov Today 4(9): 431-439). These voltage sensitivity assays are based on fluorescence resonance energy transfer (FRET) between the membrane soluble voltage sensitive dye DiSBAC2(3) and the fluorescent phospholipid CC2-DMPE attached to the outer layer of the plasma membrane and used as a FRET donor. Change. The change in membrane potential (Vm) causes the negatively charged DiSBAC2(3) to redistribute across the plasma membrane and the amount of energy transfer from CC2-DMPE changes accordingly. Monitoring changes in fluorescence emission using the VIPRTM II, an integrated liquid processor and fluorescence detector designed for cell-based screening in 96-well or 3-84-well microtiter plates. Identifying Corrector Compounds To identify small molecules that correct migration defects associated with ΔΡ508-€, a single addition of the HTS assay format was developed. The cells were incubated for 16 hours at 37 ° C in the presence or absence (negative control) of the test compound in serum-free medium. As a positive control group, cells inoculated in 384-well plates at 155839.doc •108·201204720 were incubated at 27 ° C for 16 hours to "temperature correction" ΔΡ508-ΟΡΤΙΙ. The cells were then washed 3 times with Krebs Ringers solution and loaded with a voltage sensitive dye. To activate AF508-CFTR, 10 μΜ of forskolin and CFTR potentiator genistein (20 μΜ) were added to each well along with the Cl_free medium. The addition of Cl-free medium promoted the outflow of C1· in response to AFSOS-CFTR activation, and optically monitored membrane depolarization using FRET-based voltage sensing dyes. Identification of synergist compounds To identify synergists for AF508-CFTR, a dual-addition HTS assay format was developed. During the first addition, a Cr-free medium with or without the test compound was added to each well. After 22 seconds, Cl_free medium containing 2 to 10 μ of lanolin was added again to activate AFSOS-CFTR. The extracellular Cl_ concentration after 28 additions was 28 mM, which prompted Cl_ to flow out in response to activation of AFSOS-CFTR, and optically monitored the resulting membrane depolarization using a FRET-based voltage sensing dye. Solution No. 1 electrolyte: (in mM) NaCl 160; KC1 4.5; CaCl2 2 ; MgCl2 1 ; HEPES 10, pH 7.4 (with NaOH). Gas-free ion electrolyte: The chloride salt in the No. 1 electrolyte (described above) was changed to a gluconate. CC2-DMPE: Prepared as a 10 mM stock solution in DMSO and stored at -20 °C.

DiSBAC2 (3): Prepared in 10 mM stock solution in DMSO and stored at -20 °C. 155839.doc -109- 201204720 Cell Culture Optical measurements of membrane potential were performed using NIH3T3 mouse fibroblasts stably expressing ΔF508-CFTR. In a 175 cm2 culture flask at 37 ° C in 5% CO 2 and 90% humidity, supplemented with 2 mM branic acid, 10% fetal bovine serum, sputum, β-ΜΕ, lx penicillin/streptomycin Cells were maintained in Duchenne's modified Ig's medium at 25 mM HEPES. For all optical assays, cells were seeded at 30,000 per well in 384 well matrigel-coated plates and incubated at 37 °C for 2 hours, then at 27 °C for 24 hours for synergists Detection. For corrective testing, cells were cultured for 16 to 24 hours in the presence and absence of compounds at 27 ° C or 37 ° C. An electrophysiological assay for detecting the AFSOS-CFTR regulatory properties of a compound.

Ussing chamber assay The Using chamber assay was performed on polarized epithelial cells expressing AFSOS-CFTR to further characterize the AFSOS-CFTR modulator identified in the optical assay. The FRTAF5Q8 CFTR epithelial cells grown on the Costar Snapwell cell culture insert were placed in a Ussing chamber (Physiologic Instruments, Inc., San Diego, CA) and a voltage clamp system (Department of Bioengineering, University of Iowa, IA) was used. And a single layer continuous short circuit with Physiologic Instruments, Inc., San Diego, CA. The transepithelial resistance was measured by applying a 2 mV pulse. Under these conditions, the FRT epithelium exhibits a resistance of 4 Κ Ω/cm 2 or more than 4 Κ Ω/cm 2 . The solution was maintained at 27 ° C and bubbled with air. Use cell-free inserts to correct electrode offset potential and fluid resistance. Under these conditions, the current reflects the flow rate of the AF508-CFTR exhibited by the C1·through the 155839.doc •110· 201204720 apical membrane. Isc is obtained digitally using the MP100A-CE interface and AcqKnowledge software (v3.2.6; BIOPAC Systems, Santa Barbara, CA). Identifying Corrector Compounds A typical approach utilizes a basement to apical membrane Cl_ concentration gradient. To establish this gradient, a standard Ringer's solution was used on the basolateral membrane, and the apical membrane NaCl was changed to an equivalent molar concentration of sodium gluconate (titrated to pH 7.4 with NaOH) to obtain a wide range of cr concentrations across the epithelium. gradient. All experiments were performed with a complete single layer. To fully activate ΔΡ508-ΟΡΤΚ, apply forskolin (10 μΜ) and PDE inhibitor ΙΒΜΧ (100 μΜ), followed by the CFTR synergist genistein (50 μΜ) as observed in other cell types, at low temperatures Incubating FRT cells with stable performance △ F508-CFTR will increase the functional density of CFTR in the plasma membrane. To determine the activity of the correcting compound, cells were incubated with 10 μΜ of the test compound for 24 hours at 37 ° C and then washed 3 times, followed by recording. The cAMP-mediated and genistein-mediated Isc of the compound-treated cells was corrected for the 27 ° C and 37 ° C control groups and expressed as a percentage of activity. Preincubation of cells with corrective compounds significantly increased cAMP mediated and genistein-mediated Isc compared to the 37 °C control group. Identification of Synergist Compounds A typical approach utilizes a basolateral to apical cr concentration gradient. To establish this gradient, a standard Ringer's solution was applied to the basolateral membrane and infiltrated with nystatin (360 pg/ml), and the apical membrane NaCl was changed to an equimolar concentration of sodium gluconate (using NaOH was titrated to pH 7.4) to obtain a gradient across the epithelium of 155839.doc -111 - 201204720. All experiments were performed 30 minutes after nystatin infiltration. Forskolin (10 μM) and all test compounds were added to both sides of the cell culture insert. The efficacy of the ΔF508-CFTR synergist was assumed to be compared to the efficacy of the known synergist genistein. Solution basement solution (in mM): NaCl (135), CaCl2 (1.2), MgCl2 (1.2), Κ2ΗΡ〇4 (2.4), KHP〇4 (0.6), N-2-hydroxyethylpiperazine -N'-2-ethanesulfonic acid (HEPES) (10) and dextrose (10). The solution was titrated to pH 7.4 with NaOH. Top Membrane Solution (in mM): Same as the basolateral solution, where NaCl was changed to sodium gluconate (135). Cell culture The Ussing chamber experiment was performed on the putative ΔF508-CFTR modulator identified by our optical assay using Fisher's mouse epithelial (FRT) cells expressing ΔF508-CFTR (FRTAF508-cftr). Cells were grown on a Costar Snapwell cell culture insert and at 37. 〇 and 5% c〇2 under the supplement of 5 ° /. The fetal calf serum, 100 u/ml penicillin and 1〇〇 48/1111 streptomycin were cultured for 5 days in Coon's modified Ham's F-12 medium. Cells were incubated at 27 °C for 16 to 48 hours to characterize ΔFSOS-CFTR prior to characterizing the synergist activity of the compounds. To determine the activity of the agonist compound, cells were incubated for 24 hours at 27 ° C or 37 ° C in the presence and absence of compound. Whole-cell recordings were monitored by temperature-corrected and tested using a perforated patch whole-cell recording method. Macroscopic AF508-CFTR current (IaF508) in NIH3T3 cells with a stable performance of ΔΡ508-€ΡΤΙΙ. Briefly, Iaf5〇8 was subjected to voltage box recording at room temperature using an Axopatch 200B patch clamp amplifier (Axon Instruments Inc., Foster City, CA). All records were taken at a sampling frequency of l kHz and low pass filtration was performed at 1 kHz. The pipette has a resistance of 5 ΜΩ to 6 ΜΩ when filled with an intracellular solution. Under these recording conditions, the calculated C1·reversal potential (EC1) at room temperature was -28 mV» All records had a >20 GQ seal resistance and a series resistance of &lt;15 ΜΩ. Pulse generation, data acquisition and analysis were performed using a PC equipped with a Digidata 1320 A/D interface and Clampex 8 (Axon Instruments Inc.). The cell contained &lt;250 μL of physiological saline and was continuously perfused at a rate of 2 ml/min using a gravity-driven perfusion system. Identification of the corrective compound To determine the activity of the correcting compound to increase the density of the functional AFSOS-CFTR in the plasma membrane, we used the above-described perforated patch recording technique to measure the current density after 24 hours of treatment with the correcting compound. To fully activate AF508-CFTR, 10 μΜ of forskolin and 20 μ of genistein were added to the cells. Under the conditions recorded by us, the current density after incubation at 27 ° C for 24 hours was higher than that observed after incubation at 37 ° C for 24 hours. These results are consistent with the known effects of low temperature incubation on the density of AFSOS-CFTR in the plasma membrane. To determine the effect of the correcting compound on the CFTR current density, cells were incubated with 10 μΜ of the test compound for 24 hours at 37 ° C, and the current density was compared to the 27 ° C and 37 ° C control groups (% active). . Prior to recording, the cells were washed 3 times with extracellular recording medium to remove any residual test compound 155839.doc • 113-201204720. Pre-culture with 1 〇 μΜ corrective compound significantly increased cAMP-dependent and genistein-dependent currents compared to the 37 °C control group. Identification of potentiator compounds The ability of AFSOS-CFTR potentiators to increase macroscopic AFSOS-CFTR Cl· current (ΙΔΡ508) in NIH3T3 cells stably expressing AFSOS-CFTR was also investigated using perforated patch recording techniques. Synergists identified by optical detection resulted in increased dose dependence of IAF508 with similar potency and efficacy observed in optical assays. In all cells studied, the potential was reversed to about -30 mV before and during the application of the potentiator, which is the calculated Ecl (-28 mV). Solution intracellular solution (in mM): aspartic acid planer (90), CsCl (50), MgCl2 (1), HEPES (IO) and 240 pg/ml amphotericin-B (pH adjustment with CsOH) To 7.35). Extracellular solution (in mM): N-methyl-D-glucosamine (NMDG)-Cl (150), MgCl 2 (2), CaCl 2 (2), HEPES (10) (pH adjustment to HC1 7.35). Cell Culture NIH3T3 mouse fibroblasts stably expressing AFSOS-CFTR were used in whole cell recording. In a 175 cm2 culture flask at 37 ° C in 5% CO 2 and 90% humidity, supplemented with 2 mM branic acid, 10% fetal bovine serum, 1 χΝΕΑΑ, β-ΜΕ, 1 X penicillin/chain Cells were maintained in Duchenne modified Ig's medium with 25 mM HEPES. For whole-cell recording, 2,500-5,000 cells were seeded onto poly-L-amino acid coated glass coverslips and incubated at 27 ° C for 24 to 48 hours before being used to test synergists 155839.doc -114- 201204720 Activity; and incubated at 37 ° C in the presence or absence of a corrective compound for measuring the activity of the corrective agent. Single Channel Recording The activity of the single channel activity and synergist compound of the temperature-corrected AFSOS-CFTR stably expressed in NIH3T3 cells was observed using a cut-out inner and outer membrane. Briefly, single channel activity was voltage clamped at room temperature using an Axopatch 200B patch clamp amplifier (Axon Instruments Inc.). All records were taken at a sampling frequency of 10 kHz and low pass at 400 Hz. The membrane pipette was made by Corning Kovar Sealing #7052 glass (World Precision Instruments, Inc., Sarasota, FL) and had a resistance of 5 ΜΩ to 8 ΜΩ when filled with an extracellular solution. After excision, ΔF508-CFTR was activated by the addition of 1 mM Mg-ATP and 75 nM cAMP-dependent protein kinase catalytic subunit (PKA; Promega Corp. Madison, WI). After the channel activity is stabilized, the membrane is perfused using a gravity driven microperfusion system. The influent is placed adjacent to the membrane so that solution exchange is completed in 1 to 2 seconds. To maintain AF508-CFTR activity during rapid perfusion, a non-specific phosphatase inhibitor F (10 mM NaF) was added to the electrolyte. Under these recording conditions, channel activity remained constant throughout the duration of the membrane recording (up to 60 minutes). The current generated by the positive charge moving from the intracellular solution to the extracellular solution (the anion moves in the opposite direction) is shown as a positive current. The pipette potential (Vp) was maintained at 80 mV.

Channel activity was analyzed from a membrane containing S2 active channels. The maximum number of sync openings determines the number of active channels during the course of the experiment. To determine the single-channel current amplitude, the data recorded in the 120 seconds of AF508-CFTR 155839.doc •115-201204720 activity was filtered off-line at 100 Hz and then the biofilm was used as the software (Bi〇). -L〇gic Comp. France) Constructs a full-point amplitude histogram fitted to a multi-Gaussian function. The total microscopic current and open probability (P.) were determined from the channel activity of 12 sec. P〇 is determined using biofilm software or self-relevant P'l/VN) (where 1 = average current, 丨 = single channel current amplitude and n = number of active channels in the membrane). Solution extracellular solution (in mM): NMDG (150), aspartic acid (15 〇), CaCl 2 (5), MgCl 2 (2), and HEPES (10) (pH adjusted to 7.35 by Tris) ° Intracellular solution (in mM): NMDG-C1 (150), MgCl2 (2), EGTA (5), TES (10), and Tris base (14) (pH adjusted to 7.35 with HC1). Cell Culture NIH3T3 mouse fibroblasts stably expressing AFSOS-CFTR were used for excision patch clamp recording. At 37. (: in 5% C〇2 and 90% humidity, in a 175 cm2 culture flask, supplemented with 2 mM branic acid, 1% fetal calf serum, sputum, β-ΜΕ, lx penicillin/streptococcus Cells were maintained in Duchenne's modified Ig's medium with 25 ηιΜ HEPES. For single-channel recording, 2,500 to 5,000 cells were seeded on poly-L-amino acid-coated glazed coverslips and incubated at 27 °C. 24 to 48 hours of use. The activity of Compound 2 (ECso) has been measured and not shown in Table 2-2 using the procedure described above. 155839.doc • 116· 201204720 Table 2-2 JC5O/EG50^ 4 Activity per mu ratio grading: +&lt;=25.0&lt;++&lt;=100.0&lt;+++ 4 Compound numb 4_,: maximum gong red of the classification:;, compound 2 -hH- +++ The activity of Compound 3 (i.e., EC5〇) has been measured and shown in Table 3-2 for the procedure described above. Table 3-2 IC50/EC50 Classification: '+++&lt;=2.0&lt;++ &lt;=5.0&lt;+ 3:: Γ Γ Activity percentage grading: +^^25.0^^+(=100.0^++ 4 The compound is classified by EC50 by the bee; ^gongdong compound 3 +-H- +-H- Other Embodiments All mentioned in the present invention The disclosures and patents are hereby incorporated by reference in their entirety in their entirety in the extent of the extent of the disclosure of the disclosure of the disclosure of The meaning of the terms in the patent or the disclosure is inconsistent with the meaning of the terms used in the present invention, and the meaning of the terms in the present invention is intended to be dominant. Moreover, the above discussion merely discloses and describes an exemplary embodiment of the present invention. It will be apparent to those skilled in the art that the present invention is susceptible to the scope of the invention and the scope of the invention and the scope of the invention. 155839.doc -117-

Claims (1)

201204720 VII. Patent Application Range: 1. A pharmaceutical composition comprising: a compound of formula I Or a pharmaceutically acceptable salt thereof, wherein: Ring A is selected from the group consisting of: R1 is -CF3, -CN or -C tri CCH2N(CH3)2; R2 is hydrogen, -CH3, -CF3, -OH or -CH2OH; R3 is argon, -CH3, -OCH3 or -CN; the constraint is R2 Hydrogen is different from R3; and one or both of the following: B. Compound of formula II Formula II or a pharmaceutically acceptable salt thereof, wherein: 155839.doc 201204720 T is -CH2-, -CH2CH2-, -CF2-, -c(ch3)2- or -c(o)-; R!• H, aliphatic, halo, CF3, CHF2, CHCk aliphatic); and RD1 or RD2 is ZDR9, wherein: ZD is a bond, CONH, S02NH, SC^NCCw alkyl), CH2NHS02, CH2N (CH3 ) S02, CH2NHCO, COO, S02 or CO; and R9 is H, CN6 aliphatic or aryl; and/or C. Or a pharmaceutically acceptable salt thereof, wherein: .R is hydrazine, OH, OCH3, or two R together form -0CH20- or -OCF20-; R_4 is fluorene or a hospital base; R5 is hydrazine or F; R6 is Η or CN ; R7 is Η, -(:Η2(:Η(ΟΗ)(:Η2ΟΗ, -CH2CH2N+(CH3)3 or -CH2CH2OH; R8 is Η, OH, -CH2CH(OH)CH2OH, -CH2OH, or R7 2. A pharmaceutical composition of claim 1 comprising a compound of formula I and a compound of formula 。. 3. The pharmaceutical composition of claim 1 comprising Formula I And a pharmaceutical composition according to any one of claims 1 to 4, wherein the pharmaceutical composition comprises the compound of the formula I, the compound of the formula II, and the compound of the formula III. The compound of formula I is compound 1 The pharmaceutical composition according to any one of claims 1 to 4, wherein the compound of the formula II is the compound 2 The pharmaceutical composition according to any one of claims 1 to 4, wherein the hydrazine compound is compound 3 155839.doc 201204720 8. A pharmaceutical composition comprising a component selected from any of the embodiments described in Table A, and one or both of the following: a) selected from column B of Table I a component of any of the embodiments; and/or b) a component selected from any of the embodiments described in column C of Table I. Table IA Example B Blocking Example cm Example paragraph paragraph heading paragraph heading paragraph heading II.A.1. Formula I compound II.B.1. Formula II compound II.C.1. Formula III compound II.A. 2. Compound 1 II.B.2. Compound 2 II.C.2. Compound 3 9. The use of a pharmaceutical composition according to any one of claims 1 to 8 for the manufacture of a CFTR for the treatment of humans Drugs for guiding diseases. 10. The use of claim 9, wherein the CFTR-mediated disease is selected from the group consisting of cystic fibrosis, asthma, smoking-induced COPD, chronic bronchitis, sinusitis, constipation, pancreatitis, pancreatic insufficiency, Male infertility, mild lung disease, idiopathic pancreatitis, allergic bronchi and pulmonary aspergillosis (ABPA), liver disease, hereditary emphysema, hereditary blood color caused by congenital bilateral vas deferens (CBAVD) Disease; coagulation fibrinolytic defects, such as protein C deficiency; hernia-type hereditary angioedema; lipid-deficient deficiency such as steroidal steroidal cholesterol; type 1 chylomicronemia, no beta lipoproteinemia; lysosomes Storage diseases, such as plant cell disease / pseudo-Heroler (pseudo_Hurler); mucop〇lysacchand〇ses, Sandh〇f/Taxax ( Tay-Sachs), „Kruger Na (4) Multi-knife disease/insulinemia, diabetes, Lajon's dwarfism (^ 155839.doc 201204720 dwarfism), myeloperoxidase deficiency Primary hypothyroidism, melanin , type 1 glycolytic CDG (glycanosis CDG type 1), congenital hyperthyroidism, osteogenesis imperfecta, hereditary hypoglycemia, ACT deficiency, diabetes insipidus (DI), neurophysiological DI (neurophySeal DI ), renal-derived DI, Charcot-Marie Tooth syndrome, Perlizaeus-Merzbacher disease; neurodegenerative diseases such as Alzheimer's disease (Alzheimer's disease) Disease), Parkinson's disease, amyotrophic lateral sclerosis, progressive supranuclear palsy, Pick's disease; several polyglutamate neuropathy such as Huntington's disease (Huntington's), type I spinal cerebellar dysmotility, ridge delay Zhao muscle atrophy, dentate nucleus nucleus pallidus subalkal atrophy (dentatorubal pallidoluysian) and muscular dystrophy, and spongiform encephalopathy, such as hereditary gram-Asia Creutzfeldt-Jakob disease (due to defects in prion protein treatment), Fabry disease, Straussler-Scheinker syndr Ome), COPD, dry eye or Sjogren's disease, osteoporosis, osteopenia, bone healing and bone growth (including bone repair, bone regeneration, reduction of bone resorption and increased bone deposition), Ge G〇rham, s Syndrome; gas-ion channel disease, such as congenital muscle stiffness (Thomson and Becker forms), type III Bartter's syndrome, Dent's disease (Dent's disease), hyperekplexia, epilepsy, lysosomal storage disease, Anman 155839.doc 201204720 syndrome (Angelman syndrome), and primary ciliary dyskinesia (PCD) The terminology of the structure and/or function of hereditary disorders, including PCD (also known as Kartagener syndrome) with visceral retrograde, pCD without visceral retrograde, and cilia hypoplasia. 11. The use of claim 10, wherein the CFTR-mediated disease is cystic fibrosis, COPD, emphysema, dry eye or osteoporosis. 12. The use of claim 11 wherein the CFTR-mediated disease is cystic fibrosis. 13. The use of any one of claims 9 to 12 wherein the patient has one or more of the following mutations in human CFTR: R117H&amp; G551D. 14. The use of claim 13 wherein the drug is for treating cystic fibrosis or reducing the severity of the patient having the mutation of human CFTR. 1 5 - The use of claim 13 wherein the drug is for treating or reducing the severity of cystic fibrosis in a patient having the G5 5 1D mutation of human CFTR. 16. The use of claim 14, wherein the medicament is for treating or reducing the severity of cystic fibrosis in a patient having the ΔΡ508 mutation of human CFTR on at least one of the dual genes. 17. The use of claim 14, wherein the medicament is for treating cystic fibrosis or reducing the severity of the AF508 mutation in human CFTR on two dual genes. The use of claim 15, wherein the medicament is for treating cystic fibrosis or reducing the severity of a patient having the G55丨D mutation having human CFTR on at least one of the dual genes. 19. The use of claim 15, wherein the medicament is for treating or reducing the severity of cystic fibrosis in a patient having the G55 1D mutation of human CFTR on two dual genes. a kit for measuring the activity of a CFTR or a fragment thereof in a biological sample, either in vitro or in vivo, comprising: (i) a pharmaceutical composition according to any one of claims 1 to 8; Ii) for the following instructions: a) contacting the composition with the biological sample; b) measuring the activity of the CFTR or fragment thereof. 21_ The kit of claim 20, further comprising instructions for: a) contacting other compounds with the biological sample; b) measuring the activity of the CFTR or fragment thereof in the presence of other compounds; and c) The activity of the CFTR or a fragment thereof in the presence of other compounds is compared to the activity of the CFTR or a fragment thereof in the presence of a composition comprising the pharmaceutical composition according to any one of claims 1 to 8. 22. The set of claim 21, wherein the density of the CFTR or a fragment thereof is measured by the step of comparing the activity of the CFTR or a fragment thereof. 155839.doc 201204720 IV. Designation of the representative representative: (1) The representative representative of the case is: (none) (2) The symbol of the symbol of the representative figure is simple: 5. If there is a chemical formula in this case, please reveal the best indication of the characteristics of the invention. Chemical formula: Formula I Formula II 155839.doc