Method of obtaining modulators of cystic fibrousis transmembrane conductance regulator

FIELD: chemistry.

SUBSTANCE: invention relates to field of organic chemistry, namely to method of obtaining compound of formula

1,

including condensation of carboxylic acid of formula

2

with aniline of formula

3 in presence of TZR®, where each R2 and R4 independently represents C1-6 alkyl with linear or branched chain, and each C1-6 alkyl with linear or branched chain is independently and optionally substituted with -OR'; each R5 represents OC(O)OR' or R4 and R5, taken together, form group , y represents 0, each R' represents C1-4 alkyl group, optionally substituted with one or more groups, selected from oxo and -O-C1-4-alkyl group. Invention also relates to intermediate compounds and methods of their obtaining.

EFFECT: elaborated is novel method of obtaining formula 1 compound, which can be useful as modulator of cystic fibrosis transmembrane conductance regulator (CFTR).

52 cl, 10 ex

 

The application for approval of priority

This application claims priority under three provisional patent applications U.S., having serial numbers 61/162148 filed March 20, 2009; 61/246303, filed September 28, 2009; and 61/248565, filed October 5, 2009. Each of the foregoing provisional patent applications are included in full in the present application by reference.

The technical field to which the present invention

The present invention relates to a method for modulators of transmembrane conductance regulator cystic fibrosis ("CFTR").

Background of invention

Cystic fibrosis (CF) is a recessive genetic disease that affects approximately 30,000 children and adults in the United States and approximately 30,000 children and adults in Europe. Despite progress in the treatment of CF, the method of its treatment none.

CF is caused by mutations in the gene regulator transmembrane conductance cystic fibrosis (CFTR), which encodes epithelial chloride ion channel responsible for assisting in the regulation of salt and water absorption and secretion in various tissues. Drugs based on small molecules, known as potentiate tools that increase the open probability of CFTR channel, represent a potential therapeutic Stra is egiu for the treatment of CF.

In particular, CFTR is a cAMP/ATP-mediated anion channel that is expressed in various cell types, including absorptive and secretory epithelial cells, where it regulates the flow of anions through the membrane and the activity of other ion channels and proteins. In epithelial cells, normal functioning of CFTR is critical to maintain the transport of electrolytes in the body, including the respiratory and digestive tissue. CFTR consists of approximately 1480 amino acids that encode a protein consisting of a tandem repeat transmembrane domains, each of which contains six transmembrane double helices and a nucleotide binding domain. These two transmembrane domain are linked by a large polar regulatory (R) domain with multiple phosphorylation sites that regulate channel activity and cell traffic.

The gene encoding CFTR, was identified and sequenced (See Gregory, R. J. et al. (1990) Nature 347:382-386; Rich, D. P. et al. (1990) Nature 347:358-362), (Riordan, J. R. et al. (1989) Science 245:1066-1073). The defect in this gene causes mutations in CFTR, leading to cystic fibrosis ("CF"), the most common fatal genetic disease in humans. Cystic fibrosis affects approximately one out of every 2,500 children of early age in the United States. Of the U.S. population overall, 10 million people have one copy of the defective gene without the explicit effects of the disease. In contrast, subjects with two copies of the CF associated gene suffer from debilitating and fatal effects of CF, including chronic lung disease.

In patients with CF, mutations in CFTR endogenously expressed in respiratory epithelium, leading to reduced apical anion secretion, causing an imbalance in the transport of ions and fluids. The resulting decrease in anion transport contributes to increased accumulation of mucus in the lungs associated with microbial infections that ultimately lead to the death of CF patients. In addition to respiratory disease, CF patients typically suffer from gastrointestinal problems and insufficient pancreatic function, which if untreated leads to death. In addition, most men with cystic fibrosis are infertile, and reduced fertility in women with cystic fibrosis. In contrast to the severe effects of the two copies of the CF associated gene, subjects with one copy of the CF associated gene exhibit enhanced resistance to cholera and dehydration from diarrhea - possibly explaining the relatively high prevalence of CF gene among the population.

Sequence analysis of the CFTR gene CF chromosomes revealed a number of different disease-causing mutations (Cutting, G. R. et al. (1990) Nature 346:366-369; Dean, M. et al. (1990 Cell 61:863:870; and Kerem, B-S. et al. (1989) Science 245:1073-1080; Kerem, B-S, et al. (1990) Proc. Natl. Acad. Sci. USA 87:8447-8451). Currently identified more than 1000 disease causing mutations in the CF gene (http://www.genet.sickkids.on.ca/cftr/app). The most common mutation is a deletion of phenylalanine at position 508 amino acid sequence of CFTR, and it is usually indicated as ΔF508-CFTR. This mutation occurs in approximately 70% of cases of cystic fibrosis, and it is associated with severe disease.

The deletion of residue 508 in ΔF508-CFTR prevents proper installation of the nascent protein. This leads to the inability of the mutant protein to exit the ER and move to the plasma membrane. As a result, the number of channels present in the membrane, far from determined in cells expressing wild-type CFTR. In addition to the disrupted traffic, this mutation leads to the defect of the gate mechanism of the channel. All together the reduced number of channels in the membrane and the defect of the gate mechanism, lead to a reduction of anion transport across the epithelium, leading to impaired transport of ions and fluids. (Quinton, P. M. (1990), FASEB J. 4: 2709-2727). Research, however, showed that reduced the number of ΔF508-CFTR in the membrane are functional, albeit less than wild-type CFTR. (Dalemans et al. (1991), Nature Lond. 354: 526-528; Denning et al., supra; Pasyk and Foskett (1995), J. Cell. Biochem. 270: 12347-50). In addition to ΔF508-CFTR, else C is bolovanje, causing mutations in CFTR, which leads to disrupted traffic, synthesis and/or the gate mechanism of the channel could be adjusted either by activation or down-regulation for changes in anion secretion and modification of progression and/or severity of the disease.

Although CFTR transports various molecules, in addition to anions, it is clear that this role (transport anions) represents one important element in the mechanism of transport of ions and water across the epithelium. Other elements include the epithelial Na+channel, ENaC, Na+/2Cl-/K+co-Transporter, Na+-K+-ATPase pump and basolateral membrane K+channels, which are responsible for the absorption of chlorine in the cell.

These elements work together to achieve directed transport through the epithelium through their selective expression and localization in the cell. Absorption of chlorine occurs as a result of coordinated activity of ENaC and CFTR present on the apical membrane, and Na+-K+-ATPase pump and Cl ion channels expressed on basolateral the cell surface. Secondary active transport of chlorine with luminaries side leads to accumulation of intracellular chloride, which is then passively leaves the cell through Cl-channels, leading to a vector transport. The location of Na+/2Cl-/K+/sup> co-Transporter, Na+-K+-ATPase pump and basolateral membrane K+channels on basolateral surface and CFTR on luminale side coordinates the secretion of chlorine through CFTR on luminale side. Since water will probably never itself is not actively transported, its flow through the epithelium depends on very small transepithelial osmotic gradients created by the flow of sodium and chlorine.

As discussed above, it is considered that the deletion of residue 508 in ΔF508-CFTR prevents proper installation of the nascent protein, which leads to the inability of the mutant protein to exit the ER and move to the plasma membrane. As a result, the plasma membrane contains an insufficient number of Mature protein, and the transport of chlorine in epithelial tissues is significantly reduced. Indeed, it was shown that this cellular phenomenon of defective ER processing ABC transporters using ER mechanism underlies not only the CF disease, but also a wide range of other individual and hereditary diseases.

Accordingly, there is a need for modulators of CFTR activity and compositions on their basis, which can be used to modulate the activity of the CFTR in the cell membrane of a mammal.

There is a need for methods of treatment of diseases, svannah mutation in CFTR, using such modulators of CFTR activity.

There is a need for methods of modulating CFTR activity inex vivothe cell membrane of a mammal.

There is also a need for ways to produce compounds that modulate CFTR activity.

Brief description of the invention

In General, the invention provides methods of making compounds useful as modulators of CFTR.

In one aspect the invention provides a method for obtaining compounds of formula 1

,

including the condensation of the carboxylic acid of formula 2

with an aniline of formula 3

in the presence of a condensing agent selected from the group comprising tetrafluoroborate 2-chloro-1,3-dimethyl-2-imidazole, HBTU, HCTU, 2-chloro-4,6-dimethoxy-1,3,5-triazine, HATU, HOBT/EDC and T3P®.

Each R2and R4independently selected from the group comprising hydrogen, CN, CF3, halogen, C1-6alkyl straight or branched chain, 3-12 membered cycloaliphatic group, phenyl, C5-10heteroaryl or C3-7heterocyclic group, where the specified heteroaryl or heterocyclic group contains not more than 3 heteroatoms selected from O, S or N, and each C1-6alkyl straight or branched chain, 3-12 membered cycloaliphatic group which, phenyl, C5-10heteroaryl or C3-7heterocyclic group, independently and optionally, contain not more than three substituents selected from-OR', -CF3, -OCF3, SR', S(O)R', SO2R', -SCF3, halogen, CN, -COOR', -COR, -O(CH2)2N(R')(R'), -O(CH2)N(R')(R'), -CON(R')(R'), -(CH2)2OR', -(CH2)OR', CH2CN, optionally substituted phenyl or phenoxy, -N(R')(R'), -NR'r C(O)OR', -NR'r C(O)R', -(CH2)2N(R')(R') or -(CH2)N(R')(R').

Each R5independently selected from the group comprising hydrogen, -OH, NH2CN, CHF2That other', N(R')2, -NHC(O)R', NHC(O)OR', NHSO2R', -OR', OC(O)OR', OC(O)other', OC(O)NR'2CH2OH, CH2N(R')2C(O)OR', SO2Other', SO2N(R')2or CH2NHC(O)OR'.

Or R4and R5taken together form a 5-7-membered ring containing from 0 to three heteroatoms selected from N, O or S, where the specified ring optionally contains up to three substituents R3.

Each X independently represents a bond or optionally substituted C1-6alkylidene chain, where not more than two methylene units of the group X is optionally and independently replaced by a group-CO-, -CS-, -COCO-, -CONR'-, -CONR'NR'-, -CO2-, -OCO-, -NR'r CO2-, -O-, -NR'r CONR'-, -OCONR'-, -NR NR'r', -NR'r NR'r CO-, -NR'r CO-, -S-, -SO -,- SO2-, -NR'-, -SO2NR'-, NR'r SO2- or-NR'r SO2NR'-. Each Rxindependently represents R', halogen, NO2CN, CF or OCF3.

y is an integer from 0 to 4.

Each R' is independently selected from the group comprising hydrogen or optionally substituted group selected from C1-8aliphatic group, a 3-8-membered saturated, partially unsaturated, or fully unsaturated monocyclic ring containing 0 to 3 heteroatoms, independently selected from the group comprising nitrogen, oxygen, or sulfur, or an 8-12 membered saturated, partially unsaturated, or fully unsaturated bicyclic ring system containing from 0 to 5 heteroatoms, independently selected from the group comprising nitrogen, oxygen, or sulfur; or two present R', taken together with the atom (atoms) to which they are bound, form an optionally substituted 3-12-membered saturated, partially unsaturated, or fully unsaturated monocyclic or bicyclic ring containing 0 to 4 heteroatoms, independently selected from N, O, or S.

Each R3independently represents-C1-C3alkyl, C1-C3perhalogenated, -O(C1-C3alkyl), -CF3, -OCF3, -SCF3, -F, -Cl, -Br, -COOR', -COR', -O(CH2)2N(R')(R'), -O(CH2)N(R')(R'), -CON(R')(R'), -(CH2)2OR', -(CH2)OR', optionally substituted monocyclic or bicyclic aromatic ring, optionally substituted aresult is h, optionally substituted 5-membered heteroaryl ring, -N(R')(R'), -(CH2)2N(R')(R') or -(CH2)N(R')(R').

Options embodiments of this aspect include one or more of the following features. R5independently represents-OC(O)OR', -OC(O)other', or-OC(O)N(R')2and R' is not hydrogen; at least one of R4or R2independently represents a C1-6alkyl straight or branched chain, substituted by a group-COOR' or-CON(R')(R') and R' is not hydrogen. The method also includes splitting the group-OC(O)OR', -OC(O)other', or-OC(O)N(R')2with the formation of the group-OH. The method also includes the hydrolysis of each group-COOR' or-CON(R')2with the formation of the group-COOH. The hydrolysis is carried out by treating the compounds of formula 1 alcohol solvent in the presence of a base, such as NaOH, KOH or sodium methoxide. The alcoholic solvent used in the hydrolysis reaction, is a methanol. The reaction of condensation of compounds of formula 2 and the compound of formula 3 to obtain the compounds of formula 1 is carried out in the presence of a base, such as K2CO3Et3N, NMM, pyridine or DIEA. The reaction of condensation of compounds of formula 2 and the compound of formula 3 to obtain the compounds of formula 1 is carried out in the presence of a solvent, such as EtOAc, IPAc, THF, MEK, NMP, acetonitrile, DMF or 2-METI is tetrahydrofuran. The reaction of condensation of compounds of formula 2 and the compound of formula 3 to obtain the compounds of formula 1 is carried out at a reaction temperature, which is supported approximately in the range from 10°C to 78°C, such as approximately in the range from 20°C to 30°C, approximately in the range from 40°C to 50°C and approximately in the range from 42°C to 53°C. the condensation Reaction is carried out with stirring for at least 2 hours, for example, for at least 70 hours, or for at least 3 days.

In some embodiments, embodiments, R5independently represents-OC(O)OR', -OC(O)other', or-OC(O)N(R')2and R' is not hydrogen; and each of R2and R4independently selected from the group comprising hydrogen, CF3C1-C6alkyl straight or branched chain, 3-12 membered cycloaliphatic group, or phenyl.

In some additional embodiments, embodiments, R5independently represents-OC(O)OR', and R' is not hydrogen; and each of R2and R4independently represents a C1-C6alkyl straight or branched chain or 3-12 membered cycloaliphatic group.

In some embodiments embodiment R2and R4are tert-butyl.

In another aspect the invention provides a method for obtaining compounds 27,

including:

(a) condensation of compound 26

connection 13

in the presence of EDCI, HOBT and DIEA, using DMF as solvent, where the reaction temperature is supported approximately in the range from 20°C to 30°C, and the reaction is carried out for at least 70 hours, to obtain compound 14

; and

(b) treating compound 14 using KOH in methanol.

In another aspect, the invention provides a method for obtaining compounds 28,

including:

(a) condensation of compound 26

connection 20

in the presence of HATU and DIEA, using acetonitrile as solvent, where the reaction temperature is supported approximately in the range from 40°C to 50°C, and where the reaction is carried out for at least 3 days, with the connection 21

; and

(b) treating compound 21 using NaOH in methanol.

In another aspect, the invention provides a method for obtaining compounds 34,

including:

(a) condensation of compound 26

connection 32

in the presence of T3P®and pyridine, using 2-methyltetrahydrofuran as solvent, where the reaction temperature is supported approximately in the range from 42°C to 53°C, and where the reaction is carried out for at least 2 hours, to obtain compound 33

(b) treating compound 33 using a mixture of NaOMe/MeOH 2-methyltetrahydrofuran.

In one variant embodiment, the method additionally includes the stage of formation of a suspension of compound 34 in a mixture of acetonitrile and water, where the solid form of compound 34 is converted into the Compound 34.

Variants of the embodiment of the above aspect include one or more of the following features. The method also includes the dissolution of Compound 34 in a two-phase solution of 2-methyltetrahydrofuran and 0.1 n HCl, stirred. The method also includes the selection of the organic phase of the biphasic solution. The method also includes filtering and removing solids from the organic phase. The method also includes reducing the amount of organic phase by approximately 50% using distillation. The method also includes three times the following procedure: adding MeOAc, EtOAc, PAc, t-BuOAc, tetrahydrofuran (THF), Et2O or methyl tert-butyl ether (MTBE) to the organic phase as long as the volume of the organic phase will not increase by 100%, and a reduction in the content of inorganic fillers phase by 50% using distillation. The method also includes adding MeOAc, EtOAc, IPAc, t-BuOAc, tetrahydrofuran (THF), Et2O or methyl tert-butyl ether (MTBE) to the organic phase as long as the volume of the organic phase will not increase by 100%. The method also includes heating the organic phase to the boiling temperature under reflux and maintaining a specified boiling point under reflux over a period of time of at least about 5 hours. The method also includes cooling the organic phase to a temperature in the range from -5°C to 5°C within a time period of 4.5 hours 5.5 hours.

In another aspect, the invention provides compounds obtained using any of the methods described in this application.

In another aspect the invention provides a pharmaceutical composition comprising the compound obtained by any of the methods described in this application.

In another aspect, the invention provides a method of modulating CFTR activity in a biological sample, comprising the stage of contact specified biological sample with the compound obtained by any of the methods described in this application.

In another aspect the invention also provides a method of treating or attenuating the severity of the disease in a patient, comprising the introduction of a specified patient one is omposite, defined in this application, and the specified disease selected from the group including cystic fibrosis, asthma, caused by Smoking COPD (chronic obstructive pulmonary disease), chronic bronchitis, rhinosinusitis, constipation, pancreatitis, pancreatic insufficiency cancer, male infertility caused by congenital bilateral absence of the VAS deferens (CBAVD), uncomplicated form of pulmonary disease, idiopathic pancreatitis, allergic bronchopulmonary aspergillosis (ABPA), liver disease, hereditary emphysema, hereditary hemochromatosis, coagulation-fibrinolysis deficiencies, such as protein deficiency, hereditary angioedema type 1, lipid processing, such as familial hypercholesterolaemia, chylomicronemia type 1, abetalipoproteinemia, lysosomal storage disorders, such as disease cellular inclusions/disease Deri, mucopolysaccharidosis disease Sandhof/Tay-Sachs disease criglernajjar Najjar syndrome type II, polyendocrinopathy/hyperinsulemia, diabetes, dwarfism of Larona, myeloperoxidase deficiency, primary hypoparathyroidism, melanoma, glycans CDG (congenital diseases of glycosylation type 1, congenital hyperthyroidism, imperfect osteogenesis, hereditary hypofibrinogenemia, insufficient activerow the deficient clotting time(act), diabetes insipidus (ND), insipidus neurotically diabetes insipidus neurogenic diabetes, muscular atrophy, Charcot-Marie-Toot disease Pelizaeus-Merzbacher neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, progressive supranuclear paralysis, atrophy of the Peak, some polyglutamine neurological disorders such as Huntington's disease, spinal-cerebellar ataxia type I, spinal and bulbar muscular atrophy, dentato-rubro-pallido-Lisova atrophy and myotonias dystrophy, as well as spongiform encephalopathies, such as a hereditary disease of Creutzfeldt-Jakob disease (due to defective processing of the prion protein), Fabry disease, syndrome Straussler-Sheinker, COPD (chronic obstructive pulmonary disease), dry eye syndrome, or sjögren's disease, osteoporosis, osteopenia, bone healing and bone growth (including the restoration of the bone, bone regeneration, reducing bone resorption and increasing bone deposition), the syndrome Goreme, chloride of kalapati, such as congenital myotonia form (Thomson and Becker), the syndrome Bartter type III, dent disease, hyperekplexia, epilepsy, hyperekplexia, lysosomal disease accumulation, Angelman syndrome, and primary ciliary dyskinesia (PCD), a term for nasledstvo the x disorders of the structure and/or function resisteth structures, including PCD with transposition of internal organs (also known as syndrome Addition, PCD without transposition of internal organs and ciliary aplasia.

In some embodiments embodiment the disease is a cystic fibrosis.

In another aspect the invention provides a kit for use in measuring the activity of CFTR or a fragment in the biological samplein vitroorin vivoincluding:

i. the composition comprising the compound obtained by any of the methods described in this application; and

ii. instructions for:

a. contacting the composition with the biological sample; and

b. measurement of the activity specified CFTR or fragment.

In some embodiments embodiment the kit also contains instructions for:

i. contacting an additional compound with the biological sample;

ii. measurement of the activity specified CFTR or fragment in the presence of the specified additional connections; and

iii. comparison of the activity of the CFTR in the presence of the additional compound with the density of CFTR in the presence of a composition of formula 1.

Primarily, the invention provides methods of synthesis of compounds which are useful as modulators of CFTR, with a higher yield and higher purity compared with known methods.

DETAILED description of the INVENTION

I. DEFINITION IS OF

As used in this application, the following definitions are applicable, unless otherwise indicated.

The term "ABC-Transporter" as used in this application means ABC-Transporter protein or its fragment that contains at least one binding domain, where this protein or its fragment is presentin vivoorin vitro. The term "binding domain" as used in this application, means on the domain ABC-Transporter, which can communicate with the modulator. See, for example, Hwang, T. C. et al., J. Gen. Physiol. (1998): 111(3), 477-90.

The term "CFTR" as used in this application means a transmembrane conductance regulator cystic fibrosis or mutation that is capable of regulator activity, including, but not limited to, ΔF508 CFTR and G551D CFTR (see, for example, http://www.genet.sickkids.on.ca/cftr/, for CFTR mutations).

The term "modulating" as used in this application, means an increase or decrease in the quantity that can be measured.

For the purposes of the present invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed. In addition, General principles of organic chemistry are described in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 1999, and "March''s Advanced Organic Chemistry", 5th Ed., Ed.: Smith, M. B. and March, J., John Wiley & Sons, New York: 2001, p is lnoe the contents of which are incorporated into the present application by reference.

As described in this application, the compounds of the present invention optionally can be substituted by one or more substituents, such as in the General form illustrated above or illustrated with particular classes, subclasses and species of the present invention. It should be clear that the phrase "optionally substituted" is used interchangeably with the phrase "substituted or unsubstituted". As a rule, the term "substituted", regardless of standing in front of him, the word "optional" or not, refers to the replacement of hydrogen radicals in a given structure with the radical of a specified substituent.

Unless otherwise specified, optionally substituted group may contain a substituent at each substitutable position of the group, and when more than one position in any particular structure can be substituted by more than one Deputy, selected from a specified group, such substituents may be either the same or different from each other in each position. The combination of the substituents contemplated by this invention, preferred are those which lead to the formation of a stable or chemically achievable connections.

The term "stable" as used in this application, refers to compounds, which essentially do not change who I am, being exposed to conditions that make possible the reception, detection and, preferably, their separation, purification, and use for one or more destinations, disclosed in this application. In some embodiments of the incarnation stable compound or chemically achievable connection is a such that essentially does not change during curing at a temperature of 40°C or below, in the absence of moisture or other chemically reactive conditions, for at least a week.

The term "aliphatic" or "aliphatic group", as used in this application, means a linear (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is completely saturated or which contains one or more units of unsaturation, or a monocyclic hydrocarbon or bicyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic (also specified in this application as "carbocycle", "cycloaliphatic" or "cycloalkyl"), which has a single attachment point to the rest of the molecule. Unless otherwise specified, aliphatic groups contain 1-20 aliphatic carbon atoms. In some embodiments embodiment Ali is eticheskie groups contain 1-10 aliphatic carbon atoms. In other embodiments, embodiments, aliphatic groups contain 1-8 aliphatic carbon atoms. In the following embodiments, embodiments, aliphatic groups contain 1-6 aliphatic carbon atoms, and in some embodiments the embodiment of the aliphatic groups contain 1-4 aliphatic carbon atoms. In some embodiments of the incarnation "cycloaliphatic" (or "carbocycle" or "cycloalkyl") refers to a monocyclic C3-8the hydrocarbon or bicyclic or tricyclic C8-14the hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic, that has a single attachment point to the rest of the molecule, where any individual ring in the specified bicyclic ring system has 3-7 members. Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkeline, alkyline groups and their hybrids, such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl. Suitable cycloaliphatic groups include cycloalkyl, bicyclic cycloalkyl (for example, decalin) associated bridge the communication bicycloalkyl, such as norbornyl or [2,2,2] bicyclo-octyl or associated bridge connection tricyclic groups which, such as substituted.

The term "heteroaromatics as used in this application means an aliphatic group in which one or two carbon atoms are independently replaced by one or more atoms selected from oxygen, sulfur, nitrogen, phosphorus or silicon. Heteroaromatics groups can be substituted or unsubstituted, branched or unbranched, cyclic or acyclic, and include "heterocycle", "heterocyclyl", "heterocyclizations" or "heterocyclic" group.

The term "heterocycle", "heterocyclyl", "geterotsiklicheskikh" or "heterocyclic" as used in this application, means a non-aromatic, monocyclic, bicyclic or tricyclic ring system in which one or more ring members are an independently selected heteroatom. In some embodiments, implementation of "heterocycle", "heterocyclyl", "heterocyclizations" or "heterocyclic" group contains from three to fourteen ring members, where one or more ring members are a heteroatom independently selected from the group comprising oxygen, sulfur, nitrogen or phosphorus and each ring in the system contains 3 to 7 ring members.

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

The term "unsaturated" as used in this application, means that the group contains one or more units of unsaturation.

The term "alkoxy" or "thioalkyl" used in this application, refers to an alkyl group, as defined above, attached to the main carbon chain through an oxygen atom ("alkoxy") or sulfur ("thioalkyl").

The terms "halogenations and halogenoalkane" means an aliphatic or alkoxy, depending on the specific case substituted by one or more of atomate halogen. The term "halogen" or "halo" means F, Cl, Br or I. Examples halogenations groups include-CHF2, -CH2F, -CF3, -CF2or perhalogenated, such as, -CF2CF3.

The term "aryl" used alone or as part of a larger group, as in "aralkyl", "arakaki" or "aryloxyalkyl", refers to monocyclic, bicyclic and tricyclic Coliseu system containing generally from five to fourteen ring members, where at least one number of the TSO in the system is aromatic, and where each ring in the system contains 3 to 7 ring members. The term "aryl" can be used interchangeably with the term "aryl ring". The term "aryl" also refers to a heteroaryl ring systems defined in this application below.

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

Aryl (including aralkyl, Alcoxy, aryloxyalkyl and the like) or heteroaryl (including heteroalkyl, heteroaromatics and the like) group may contain one or more substituents. Suitable substituents on the unsaturated carbon atom aryl or heteroaryl group selected from a halogen; R0; -OR0; -SR0; 1,2-methylene-dioxy; 1,2-Ethylenedioxy; phenyl (Ph) optionally substituted by a group R0; -O(Ph), neobyazatel is substituted by a group R 0; -(CH2)1-2(Ph), optionally substituted by a group R0; -CH=CH(Ph), optionally substituted by a group R0; -NO2; -CN; -N(R0)2; -NR0C(O)R0; -NR0C(O)N(R0)2; -NR0CO2R0; -NR0NR0C(O)R0; -NR0NR0C(O)N(R0)2; -NR0NR0CO2R0; -C(O)C(O)R0; -C(O)CH2C(O)R0; -CO2R0; -C(O)R0; -C(O)N(R0)2; -OC(O)N(R0)2; -S(O)2R0; -SO2N(R0)2; -S(O)R0; -NR0SO2N(R0)2; -NR0SO2R0; -C(=S)N(R0)2; -C(=NH)-N(R0)2; or -(CH2)0-2NHC(O)R0where each independent case R0selected from hydrogen, optionally substituted C1-6aliphatic group, an unsubstituted 5-6 membered heteroaryl or heterocyclic ring, phenyl, -O(Ph), or-CH2(Ph), or, notwithstanding the definition above, two independent members present in the group R0at the same Deputy or different substituents, taken together with the atom(s) to which each R0group, form a 3-8-membered cycloalkyl, heterocyclyl, aryl or heteroaryl ring containing 0-3 heteroatoms independently selected from the group comprising nitrogen, oxygen or sulfur. Optional substituents in aliphatics the Oh group, R 0selected from NH2, NH(C1-4aliphatic group), N(C1-4aliphatic group)2, halogen, C1-4aliphatic, OH, O(C1-4aliphatic group), NO2CN, CO2H, CO2(C1-4aliphatic group), - O(halogen C1-4aliphatic group) or a halogen C1-4aliphatic group, where each of the above C1-4aliphatic groups of R0is unsubstituted.

Aliphatic or heteroaromatics group or non-aromatic heterocyclic ring may contain one or more substituents. Suitable substituents on the saturated carbon of an aliphatic or heteroaromatics group or non-aromatic heterocyclic ring are selected from those listed above for the unsaturated carbon aryl or heteroaryl group and additionally include the following: =O, =S, =NNHR*, =NN(R*)2, =NNHC(O)R*, =NNHCO2(alkyl), =NNHSO2(alkyl), or =NR*, where each R* is independently selected from the group comprising hydrogen or optionally substituted C1-6aliphatic group. Optional substituents in the aliphatic group of R* are selected from NH2, NH(C1-4aliphatic group), N(C1-4aliphatic group)2, halogen, C1-4aliphatic group, OH, O(C1-4aliphatic group), NO2CN, CO2H, CO2(C1-4Alif the political group), O(halogen C1-4aliphatic group) or a halogen(C1-4aliphatic group), where each of the above C1-4aliphatic groups of R* is unsubstituted.

Optional substituents on the nitrogen non-aromatic heterocyclic ring are selected from-R+, -N(R+)2, -C(O)R+, -CO2R+, -C(O)C(O)R+, -C(O)CH2C(O)R+, -SO2R+, -SO2N(R+)2, -C(=S)N(R+)2, -C(=NH)-N(R+)2or-NR+SO2R+; where R+represents hydrogen, optionally substituted C1-6aliphatic group, optionally substituted phenyl, optionally substituted-O(Ph), optionally substituted-CH2(Ph), optionally substituted -(CH2)1-2(Ph); optionally substituted-CH=CH(Ph); or an unsubstituted 5-6 membered heteroaryl or heterocyclic ring containing from one to four heteroatoms, independently selected from the group comprising oxygen, nitrogen or sulfur, or, notwithstanding the definition above, two independent members present in the group R+at the same Deputy or different substituents, taken together with the atom(s) to which each R+group, form a 3-8-membered cycloalkyl, heterocyclyl, aryl or heteroaryl ring containing 0-3 heteroatoms independently wybranych group, including nitrogen, oxygen, or sulfur. Optional substituents in the aliphatic group or the phenyl ring of R+selected from NH2, NH(C1-4aliphatic group), N(C1-4aliphatic group)2, halogen, C1-4aliphatic group, OH, O(C1-4aliphatic group), NO2CN, CO2H, CO2(C1-4aliphatic group), - O(halogen C1-4aliphatic group) or a halogen(C1-4aliphatic group), where each of the above C1-4aliphatic groups of R+is unsubstituted.

The term "alkylidene chain" refers to a straight or branched carbon chain, which may be fully saturated or may contain one or more units of unsaturation and contains two points of connection to the rest of the molecule. The term "spirocyclohexane" refers to a carbocyclic ring, which may be fully saturated or may contain one or more units of unsaturation, and has two attachment points from the same ring carbon atom to the rest of the molecule.

The term "suspension" as used in this application, is defined as a mixture comprising solid and liquid, where the solid substance is not more than partially soluble in the liquid. The term "suspended" or "WM is andromania", as used in this application (example, "solid product suspended within 24 hours"), is defined as an activity aimed at the formation of the slurry, and mixing the specified suspension for a certain period of time.

The term "protective group" (PG) as used in this application means a group designed to protect a functional group, such as, for example, alcohol, amine, carboxyl, carbonyl, etc. against undesirable reactions during the procedure of synthesis. Traditionally used protective groups are disclosed in Greene and Wuts, Protective Groups in Organic Synthesis, 3rd Edition (John Wiley & Sons, New York, 1999), which is incorporated into the present application by reference. Examples of the nitrogen-protecting groups include acyl, aroline or karamelnye groups, such as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl, 2-chloroacetyl, 2-bromoacetyl, TRIFLUOROACETYL, trichloroacetyl, phthalyl, o-nitrophenoxyacetic, α-chlorobutyryl, benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl and the chiral auxiliary groups, such as protected or unprotected D, L or D, L-amino acids such as alanine, leucine, phenylalanine and the like; sulfonylurea groups, such as benzazolyl, p-toluensulfonyl and the like; urethane groups, such as benzyloxycarbonyl, p-chlorobenzenesulfonyl, p-methoxybenzenesulfonyl, p-n is traverselistener, 2-nitrobenzenesulfonyl, p-bromobenzyloxycarbonyl, 3,4-dimethoxyphenylacetone, 3,5-dimethoxybenzoquinone, 2,4-dimethoxybenzoquinone, 4-methoxybenzenesulfonyl, 2-nitro-4,5-dimethoxybenzonitrile, 3,4,5-trimethoxybenzylamine, 1-(p-biphenylyl)-1-methylethanolamine, α,α-dimethyl-3,5-dimethoxybenzoquinone, benzylaminocarbonyl, tert-butyloxycarbonyl, diisopropylperoxydicarbonate, isopropoxycarbonyl, etoxycarbonyl, methoxycarbonyl, allyloxycarbonyl, 2,2,2,-trichlorocyanuric, phenoxycarbonyl, 4-nitrophenoxyacetic, fluorenyl-9-methoxycarbonyl, cyclopentanecarbonyl, adamantanecarbonyl, cyclohexyloxycarbonyl, phenylthiocarbamyl and such, arylalkyl groups such as benzyl, triphenylmethyl, benzyloxyethyl and the like and silyl groups such as trimethylsilyl and the like. Another example of the N-protective group is tert-butyloxycarbonyl (Boc).

Examples of useful protective groups for acids are substituted alkalemia esters such as 9-fluorenylmethyl, methoxymethyl, methylthiomethyl, tetrahydropyranyloxy, tetrahydropyranyloxy, methoxyethoxymethyl, 2-(trimethylsilyl)ethoxymethyl, benzyloxyethanol, pivaloyloxymethyl, phenylacetonitrile, triisopropylchlorosilane, cinematology, aretology,finally, substituted peacelove esters, 2,2,2-trichlorethylene, 2-halogenations, ω-chloralkali, 2-(trimethylsilyl)ethyl, 2-methylthioethyl, tert-butyl, 3-methyl-3-pentalogy, dicyclopentadienyl, cyclopentyloxy, cyclohexyloxy, allyl, metalloy, cinnamony, phenyl, Silovye esters, benzyl and substituted benzyl esters, 2,6-dialkylphenol esters such as pentafluorophenyl, 2,6-dialkylanilines. Other protective groups for acids represent a methyl or ethyl esters.

How to add (method commonly referred to as "protection"), and delete (method commonly referred to as "removing protection") of such protective groups for the amine and acid are well known in the art and are available, for example, see P. J. Kocienski, Protecting Groups, Thieme, 1994, which is incorporated into the present application by reference in its entirety, and Greene and Wuts, Protective Groups in Organic Synthesis, 3rd Edition (John Wiley & Sons, New York, 1999).

Examples of suitable solvents that can be used in the present invention include, but are not limited to, water, methanol, dichloromethane (DHM), acetonitrile, dimethylformamide (DMF), methyl acetate (MeOAc), ethyl acetate (EtOAc), isopropylacetate (IPAc), tert-butyl acetate (t-BuOAc), isopropyl alcohol (IPA), tetrahydrofuran (THF), methyl ethyl ketone (MEK), tert-butanol, diethyl ether (Et 2O), methyl tert-butyl ether (MTBE), 1,4-dioxane and N-organic (NMP).

Examples of suitable binding agents that can be used in the present invention include, but are not limited to, the hydrochloride of 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide (EDCI), hexaflurophosphate 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium (HBTU), 1-hydroxybenzotriazole (HOBT), hexaflurophosphate 2-(1H-7-asobancaria-1-yl)-1,1,3,3-tetramethylurea (HATU), tetrafluoroborate 2-chloro-1,3-dimethyl-2-imidazole, 1-H-benzotriazole-1-[bis(dimethylamino)methylene]-5-changecipherspec (HCTU), 2-chloro-4,6-dimethoxy-1,3,5-triazine and 2-papapostolou anhydride (T3P®).

Examples of suitable bases that can be used in the present invention include, but are not limited to, potassium carbonate (K2CO3), N-methylmorpholine (NMM), triethylamine (Et3N; TEA), diisopropylethylamine (i-Pr2EtN; DIEA), pyridine, potassium hydroxide (KOH), sodium hydroxide (NaOH) and sodium methoxide (NaOMe; NaOCH3).

In some embodiments, embodiment two independently present in the group R0shown in the structure below, taken together with the atom(atoms) with which they are associated, with the formation of a 3-8-membered cycloalkyl, heterocyclyl, aryl or heteroaryl ring containing 0-3 heteroatoms independently selected from the group comprising nitrogen, oxygen or sulfur. Example the rings, which is formed by two independently audience groups R0taken together with the atom(atoms) with which they are associated, include, but are not limited to, the following: a) two independent members present in the group R0that are linked to the same atom and are taken together with that atom to form a ring, for example, N(R0)2where both are present in the group R0taken together with the nitrogen atom to form piperidine-1-ilen, piperazine-1-ilen, or morpholine-4-ilen group; and b) two independent members present in the group R0that are associated with different atoms and are taken together with both of those atoms to form a ring, for example where a phenyl group is substituted with two independently audience groups OR0

These two independently present in the group R0taken together with the oxygen atoms to which they are linked, with the formation of a condensed 6-membered oxygen-containing ring:

It should be clear that various other rings can be formed when two independent participants of the group R0taken together with the atom(atoms) to which each variable is bound and that the examples detailed above are not limiting.

The substituents in the rings, for example, mono - and polyarylenes, al is factual, heteroaromatics ring systems may be attached in any position of the ring, joining substituent which is chemically possible.

If not specified, it is assumed that the patterns presented in this application, also include all isomeric (e.g., enantiomeric, diastereomeric and geometric (or conformational)) forms of the structures; for example, R and S configurations for each asymmetric center, (Z) and (E) isomers on double bond, and (Z) and (E) conformational isomers. Therefore, single stereochemical isomers as well as enantiomeric, diastereomeric and geometric (or conformational) of a mixture of compounds of the present invention is included in the scope of the present invention. Unless otherwise stated, all tautomeric forms of the compounds of the present invention is included in the scope of the present invention. For example, when Rx-X in the compound of formula 1 represents hydrogen, this compound of formula 1 can exist in the form of tautomers:

Also, it is implied, if not stated otherwise, the patterns presented in this application include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having presented the structure, the exception is the group of substitution of hydrogen by deuterium or tritium, or substitution of carbon carbon13C or14C, included in the scope of the present invention. Such compounds are useful, for example, as analytical tools, probes in biological assays or as therapeutic agents.

II. The METHODS of the PRESENT INVENTION

In General, the invention provides methods of synthesis of compounds which are useful as modulators of CFTR.

In some embodiments the embodiment of the invention provides a method of obtaining compounds having the structure

In some embodiments the embodiment of the invention provides a method of obtaining compounds having the structure

In some embodiments the embodiment of the invention provides a method of obtaining compounds having the structure

In one aspect, the invention provides a method for obtaining compounds of formula 1,

including the condensation of the carboxylic acid of formula 2

with an aniline of formula 3

in the presence of a condensing agent selected from the group comprising tetrafluoroborate 2-chloro-1,3-dimethyl-2-imidazole, HBTU, HCTU, 2-chloro-4,6-dimethoxy-1,3,5-triazine, HATU, HOBT/EDC and T3P®.

Each R2and R4nez the performance of selected from the group including hydrogen, CN, CF3, halogen, C1-6alkyl straight or branched chain, 3-12 membered cycloaliphatic group, phenyl, C5-10heteroaryl or C3-7heterocyclic group, where the specified heteroaryl or heterocyclic group contains not more than 3 heteroatoms selected from O, S or N, and each C1-6alkyl straight or branched chain, 3-12 membered cycloaliphatic group, phenyl, C5-10heteroaryl or C3-7heterocyclic group is independently and optionally contains up to three substituents selected from-OR', -CF3, -OCF3, SR', S(O)R', SO2R', -SCF3, halogen, CN, -COOR', -COR, -O(CH2)2N(R')(R'), -O(CH2)N(R')(R'), -CON(R')(R'), -(CH2)2OR', -(CH2)OR', CH2CN, optionally substituted phenyl or phenoxy, -N(R')(R'), -NR'r C(O)0R', -NR'C(O)R', -(CH2)2N(R')(R') or -(CH2)N(R')(R').

Each R5independently selected from the group comprising hydrogen, -OH, NH2CN, CHF2That other', N(R')2, -NHC(O)R', NHC(O)OR', NHSO2R', -OR', OC(O)OR', OC(O)other', OC(O)NR'2CH2OH, CH2N(R')2C(O)OR', SO2Other', SO2N(R')2or CH2NHC(O)OR'.

Or R4and R5taken together, form a 5-7 membered ring containing 0-3 heteroatoms selected from N, O or S, where the specified ring optionally contains up to three substituents R3.

1-6alkylidene chain, where up to two methylene units of X are not necessarily and independently replaced by a group-CO-CS-, -COCO-, -CONR'-, -CONR'NR'-, -CO2-, -OCO-, -NR'r CO2-, -O-, -NR'r CONR'-, -OCONR'-, -NR NR'r', -NR'r NR'r CO-, -NR'r CO-, -S-, -SO -,- SO2-, -NR'-, -SO2NR'-, NR'r SO2- or-NR'r SO2NR'-.

Each Rxindependently represents R', halogen, NO2CN, CF3or OCF3, y is an integer having a value of 0-4. Each R' is independently selected from hydrogen or optionally substituted group selected from C1-8aliphatic group, a 3-8-membered saturated, partially unsaturated, or fully unsaturated monocyclic ring containing 0-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an 8-12 membered saturated, partially unsaturated, or fully unsaturated bicyclic ring system containing 0-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur; or two present group R', taken together with the atom (atoms) to which they are bound, form an optionally substituted 3-12-membered saturated, partially unsaturated, or fully unsaturated monocyclic or bicyclic ring containing 0-4 heteroatoms, independently selected from N, O, or S.

Each R3independently represents-C1-3alkyl, C1-3perhalogenated, -O(C1-3Alki is), -CF3, -OCF3, -SCF3, -F, -Cl, -Br, or-COOR', -COR', -O(CH2)2N(R')(R'), -O(CH2)N(R')(R'), -CON(R')(R'), -(CH2)2OR', -(CH2)OR', optionally substituted monocyclic or bicyclic aromatic ring, optionally substituted arylsulfonyl, optionally substituted 5-membered heteroaryl ring, -N(R')(R'), -(CH2)2N(R')(R') or -(CH2)N(R')(R').

In one variant embodiment, R5independently represents-OC(O)OR', -OC(O)other', or-OC(O)N(R')2and R' is not hydrogen. In some cases, R5represents-OC(O)OR', and R' is not hydrogen. In other cases, R5represents-OC(O)other', and R' is not hydrogen. In some other cases, R5represents-OC(O)N(R')2and R' is not hydrogen.

In one variant embodiment, the method also includes splitting-OC(O)OR', -OC(O)other', or-OC(O)N(R')2R5the group with the formation of-OH. Cleavage is carried out by treatment of compounds of formula 1 containing-OC(O)OR', -OC(O)other', or-OC(O)N(R')2R5group, an alcohol solvent in the presence of a base, such as NaOH, KOH or sodium methoxide. The alcoholic solvent used in the cleavage reaction, represents methanol, ethanol, isopropyl alcohol or tert-butanol.

In another variant embodiment, at least one of R4or R independently represents a C1-6alkyl straight or branched chain, substituted by a group-COOR' or-CON(R')2and R' is not hydrogen. In some cases, one of R4or R2is a-COOR' and R' is not hydrogen. In other cases, one of R4or R2represents-CON(R')2and R' is not hydrogen.

In one variant embodiment, the method also includes the hydrolysis of the group-COOR' or-CON(R')2at least one of R4and R2. The hydrolysis is carried out by treating the compounds of formula 1 containing-COOR' or-CON(R')2group, at least one of R4and R2using an alcoholic solvent, in the presence of a base, such as NaOH, KOH or sodium methoxide. The alcoholic solvent used in the hydrolysis, represents methanol, ethanol, isopropyl alcohol or tert-butanol.

In another variant embodiment, at least one of R4or R2independently represents a C1-6alkyl straight or branched chain, substituted by a group-COOR' or-CON(R')2and R5independently represents-OC(O)OR', -OC(O)other', or-OC(O)N(R')2and each R' is not hydrogen.

In one variant embodiment, the method also includes the hydrolysis of the group-COOR' or-CON(R')2at least one is from R 4and R2and splitting-OC(O)OR', -OC(O)other', or-OC(O)N(R')2R5group. The reaction of hydrolysis/cleavage is carried out by treatment of compounds of formula 1 containing-COOR' or-CON(R')2group, at least one of R4and R2and-OC(O)OR', -OC(O)other', or-OC(O)N(R')2R5the group, using an alcoholic solvent, in the presence of a base, such as NaOH, KOH or sodium methoxide. The alcoholic solvent used in the hydrolysis/cleavage represents methanol, ethanol, isopropyl alcohol or tert-butanol.

In another variant embodiment, the combination of a carboxylic acid of formula 2 and aniline of formula 3 is carried out in the presence of a base, such as K2CO3Et3N, N-methylmorpholine (NMM), pyridine or DIEA.

In another variant embodiment, the combination of a carboxylic acid of formula 2 and aniline of formula 3 is carried out in the presence of pyridine or DIEA.

In the following variant embodiment, the combination of a carboxylic acid of formula 2 and aniline of formula 3 is carried out in the presence of a solvent, such as EtOAc, IPAc, THF, MEK, NMP, acetonitrile, DMF or 2-methyltetrahydrofuran.

In the following variants embodiment, the condensation of the carboxylic acid of formula 2 and aniline of formula 3 is carried out at a reaction temperature that is maintained within the range from 10°C to 78°C, such as closer is Ino in the range from 20°C to 30°C, approximately in the range from 40°C to 50°C and approximately in the range from 42°C to 53°C.

In some embodiments embodiment, the condensation reaction is carried out with stirring for at least 2 hours, for example for at least 8 hours, for at least 70 hours, or for at least 3 days.

In another variant embodiment, y is set to 0.

In the following embodiments, embodiments, R2represents tert-butyl.

In some embodiments embodiment R5independently represents-OC(O)OR', -OC(O)other', or-OC(O)N(R')2and R' is not hydrogen; and each of R2and R4independently selected from hydrogen, CF3C1-C6the alkyl straight or branched chain, 3-12 membered cycloaliphatic group, or phenyl.

In some embodiments embodiment R5independently represents-OC(O)OR', -OC(O)other', or-OC(O)N(R')2and R' is not hydrogen; and each of R2and R4independently selected from C1-C6the alkyl straight or branched chain.

In some embodiments embodiment R5independently represents-OC(O)OR', -OC(O)other', or-OC(O)N(R')2and R' is not hydrogen; and each of R2and R4independently selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentile or n-hexyl.

In some the variants of embodiment R 2and R4are tert-butyl.

In one variant embodiment, the invention provides a method for obtaining compounds of formula 5

by reacting the compounds of formula 6

with a reagent capable of causing adherence of the protective group to the phenolic oxygen of compounds of formula 6 in the presence of a solvent, obtaining, thus, the compounds of formula 7,

which is subjected to nitration to form compounds of formula 8

then restore with obtaining the compounds of formula 5, where PG represents a protective group, and R4and R5have the meaning given above.

In one variant embodiment, the solvent used in the conversion of compounds of formula 6 to the compound of formula 7 represents diethyl ether or methylene chloride.

In another variant embodiment, the solvent used in the reaction for introducing the protective group represents methylene chloride.

In the following variant embodiment, PG is propoxyphenyl, methanesulfonyl, 4-nitrobenzoyl, taxiformis, butoxypropyl, tert-butoxypropyl, isopropoxyphenyl or methoxypropyl.

In another variant embodiment, PG is with the battle methoxypropyl.

In another variant embodiment, the compound of formula 7 powerhaul the nitration with a mixture of sulfuric acid, nitric acid and methylene chloride.

In one variant embodiment, nitrosoaniline formula 8 is purified by means of crystallization.

In the following variant embodiment, nitrosoaniline formula 8 is purified by means of crystallization with the use of hexane.

In another variant embodiment, the method also involves the step of contacting the compound of formula 4

with a water solution of acid to obtain the compounds of formula 2.

In one variant embodiment, the compound of formula 3 is a compound of formula 40

In another variant embodiment, the method also involves the step of contacting the compounds of formula 41

with (MTDA)

obtaining the compounds of formula 42

In the following variant embodiment, the method includes a step of recovery of the compounds of formula 42 with obtaining the compounds of formula 40.

In one variant embodiment, the compound of formula 3 is a compound of formula 43

In the following variant embodiment, the method includes article is Dios contacting the compounds of formula 44

with (MTDA)

obtaining the compounds of formula 45

In the following variant embodiment, the method includes a step of recovery of the compounds of formula 45 with obtaining the compounds of formula 43.

In another aspect, the invention provides a method for obtaining compounds of formula 2

comprising contacting the compounds of formula 4

with the aqueous acid solution, where each X independently represents a bond or is an optionally substituted C1-6alkylidene chain, where up to two methylene units of X are not necessarily and independently replaced by a group-CO-, -CS-, -COCO-CONR'-, -CONR'NR'-, -CO2-, -OCO-, -NR'r CO2-, -O-, -NR'r CONR'-, -OCONR'-, -NR NR'r', -NR'r NR'r CO-, -NR'r CO-, -S-, -SO -,- SO2-, -NR'-, -SO2NR'-, NR'r SO2- or-NR'r SO2NR'-;

each Rxindependently represents R', halogen, NO2CN, CF3or OCF3;

y is an integer having a value of 0-4; and

each R' is independently selected from the group comprising hydrogen or optionally substituted group selected from C1-8aliphatic group, a 3-8-membered saturated, partially unsaturated, or fully unsaturated monocyclic olza, containing 0-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an 8-12 membered saturated, partially unsaturated, or fully unsaturated bicyclic ring system containing 0-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur; or two present group R', taken together with the atom (atoms) to which they are bound, form an optionally substituted 3-12-membered saturated, partially unsaturated, or fully unsaturated monocyclic or bicyclic ring containing 0-4 heteroatoms independently selected from N, O, or S.

In one variant embodiment of this aspect, the compound of formula 4

obtained by contacting the compounds of formula 50

with a compound of formula 51

where RA, RBand RCcan be a C1-6alkyl.

In one variant embodiment of this aspect, the compound of formula 50 and the connection formula 50 is subjected to interaction at a temperature of from about 100°C to about 300°C. in Another variant embodiment, the compound of formula 50 and the connection formula 50 is subjected to interaction at a temperature of about 100°C. in Another variant embodiment, the compound of formula 50 and the connection formula 50 is subjected to interaction with temperaturescale 250°C. In another additional embodiment, embodiment, the compound of formula 50 and the connection formula 50 is subjected to interaction at a temperature of about 100°C and then at a temperature of about 250°C.

One additional variant embodiment of this aspect, y is set to 0.

In another aspect, the invention provides a method for obtaining compounds of formula 40

including the stage of contacting the compounds of formula 41

with (MTDA)

obtaining the compounds of formula 42

where each R2independently selected from the group comprising hydrogen, CN, CF3, halogen, C1-6alkyl straight or branched chain, 3-12 membered cycloaliphatic group, phenyl, C5-10heteroaryl or C3-7heterocyclic group, where the specified heteroaryl or heterocyclic group contains not more than 3 heteroatoms selected from O, S or N, and each C1-6alkyl straight or branched chain, 3-12 membered cycloaliphatic group, phenyl, C5-10heteroaryl or C3-7heterocyclic group is independently and optionally contains up to three substituents selected from-OR', -CF3, -OCF3, SR', S(O)R', SO2R', -SCF3, halogen, CN, -COOR', -COR, -O(CH2)2N(R')(R'), -O(CH2)N(R')(R'), -CON(R')(R'), -(CH2)2OR', -(CH2)OR', CH2CN, optionally substituted phenyl or phenoxy, -N(R')(R'), -NR'r C(O)OR', -NR'r C(O)R', -(CH2)2N(R')(R') or -(CH2)N(R')(R');

each R5independently selected from hydrogen, -OH, NH2CN, CHF2That other', N(R')2, -NHC(O)R', NHC(O)OR', NHSO2R', -OR', OC(O)OR', OC(O)other', OC(O)NR'2CH2OH, CH2N(R')2C(O)OR', SO2Other', SO2N(R')2or CH2NHC(O)OR'; and

each R' is independently selected from hydrogen or optionally substituted group selected from C1-C8aliphatic group, a 3-8-membered saturated, partially unsaturated, or fully unsaturated monocyclic ring containing 0-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an 8-12 membered saturated, partially unsaturated, or fully unsaturated bicyclic ring system containing 0-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur; or two audience groups R', taken together with the atom (atoms) to which they are bound, form an optionally substituted 3-12-membered saturated, partially unsaturated, or fully unsaturated monocyclic or bicyclic ring containing 0-4 heteroatoms, independently selected from N, O, or S.

In one variant embodiment of this aspect, the method includes hundred of the Oia recovery of the compounds of formula 42 with obtaining the compounds of formula 40.

In another aspect, the invention provides a method for obtaining compounds of formula 43

including the stage of contacting the compound having formula 44

with (MTDA)

obtaining the compounds of formula 45

where each R2independently selected from the group comprising hydrogen, CN, CF3, halogen, C1-6alkyl straight or branched chain, 3-12 membered cycloaliphatic group, phenyl, C5-10heteroaryl or C3-7heterocyclic group, where the specified heteroaryl or heterocyclic group contains not more than 3 heteroatoms selected from O, S or N, and each C1-6alkyl straight or branched chain, 3-12 membered cycloaliphatic group, phenyl, C5-10heteroaryl or C3-7heterocyclic group is independently and optionally contains up to three substituents selected from-OR', -CF3, -OCF3, SR', S(O)R', SO2R', -SCF3, halogen, CN, -COOR', -COR, -O(CH2)2N(R')(R'), -O(CH2)N(R')(R'), -CON(R')(R'), -(CH2)2'OR'-(CH2)OR', CH2CN, optionally substituted phenyl or phenoxy, -N(R')(R'), -NR'r C(O)OR', -NR'r C(O)R', -(CH2)2N(R')(R') or -(CH2)N(R')(R'); and each R' is independently selected from hydrogen or optional is entrusted substituted group, selected from C1-8aliphatic group, a 3-8-membered saturated, partially unsaturated, or fully unsaturated monocyclic ring containing 0-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an 8-12 membered saturated, partially unsaturated, or fully unsaturated bicyclic ring system containing 0-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur; or two audience groups R', taken together with the atom (atoms) to which they are bound, form an optionally substituted 3-12-membered saturated, partially unsaturated, or fully unsaturated monocyclic or bicyclic ring containing 0-4 heteroatoms, independently selected from N, O, or S.

In one variant embodiment of this aspect, the method includes a step of recovery of the compounds of formula 45 with obtaining the compounds of formula 43.

In some specific embodiments embodiment, a method of obtaining a connection 27

includes:

(a) interaction of compound 26

connection 13

in the presence of EDCI, HOBT and DIEA, using DMF as solvent, where the reaction temperature is supported approximately in the range from 20°C to 30°C, and the reaction is carried out for, at the ore, 70 hours, to obtain compound 14

; and

(b) treating compound 14 using KOH in methanol.

In another specific embodiment, embodiment, the method of obtaining connection 28

includes:

(a) interaction of compound 26

connection 20

in the presence of HATU and DIEA, using acetonitrile as solvent, where the reaction temperature is supported approximately in the range from 40°C to 50°C, and where the reaction is carried out for at least 3 days, with the connection 21

; and

(b) treating compound 21 using NaOH in methanol.

In another specific embodiment, embodiment, the method of obtaining connection 34

includes:

(a) interaction of compound 26

connection 32

in the presence of T3P® and pyridine, using a 2-methyltetrahydrofuran as solvent, where the reaction temperature maintained within the range of from about 42°to about 3°C, and where the reaction is carried out for at least 2 hours, to obtain compound 33

; and

(b) treating compound 33 using NaOMe/MeOH 2-methyltetra is hydrofuran.

In another variant embodiment, the method also includes a step of formation of a suspension of compound 34 in a mixture of acetonitrile and water, where the solid form of compound 34 is converted into the Compound 34.

In one variant embodiment, the ratio of acetonitrile to water in the suspension is about 9:1.

In another variant embodiment, the suspension is heated to a temperature approximately in the range from 73°C to 83°C.

In another variant embodiment, the connection 34 is in suspension for at least about 3 hours.

In the following variant embodiment, the method comprises quenching the reaction mixture with the aid of 1H. HCl solution; the addition of 0.1 n HCl to the mixture, thereby creating a two-phase mixture; mixing a two-phase mixture; the selection of the organic phase from the specified two-phase mixture; filtering and removing solids from the specified organic phase; reducing the volume of the organic phase by approximately 50% using distillation; implementation in three repetitions of the following steps: addition of acetonitrile to the organic phase as long as the volume of the specified organic phase will not increase by 100%, and the decrease in the volume of the organic phase by approximately 50%; the increase in the volume of the organic phase by approximately 100% by adding acetonitrile and then adding water, with the formation of suspen the AI, where the final ratio of solvent acetonitrile/water is 9:1; heating the specified suspension to a temperature approximately in the range from 73°C to 83°C; mixing the specified suspension for at least 5 hours; and cooling the specified suspension to a temperature approximately in the range from -5°C to 5°C.

In an alternative embodiment, the method comprises quenching the reaction mixture with the aid of 1.2 n HCl solution; thereby creating a two-phase mixture; mixing the specified two-phase mixture; the selection of the organic phase from the specified two-phase mixture; adding a 0.1 n HCl solution to the organic layer, thereby creating a two-phase mixture; mixing the specified two-phase mixture; the selection of the organic phase, filtering and removing solids from the specified organic phase; reducing the volume of the organic phase by approximately 50% using distillation; implementation in three repetitions of the following steps: addition of acetonitrile to the organic phase until as the volume of the specified organic phase will not increase by 100%, and the decrease in the volume of the organic phase by approximately 50%; the increase in the volume of the organic phase by approximately 100% by adding acetonitrile and then adding water, with formation of a suspension, where the final ratio of solvent acetonitrile/in what amounts to 9:1; the heating of the specified suspension to a temperature approximately in the range from 73°C to 83°C; mixing the specified suspension for at least 5 hours; and cooling the specified suspension to a temperature approximately in the range from 20°C to 25°C, filtering and removing solids from the specified slurry; washing the solid with acetonitrile having a temperature approximately in the range from 20°C to 25°C four times; and drying the solids under vacuum at a temperature from 45°C to about 55°C.

In one variant embodiment, the volume of 1H. HCl solution used for quenching the reaction is 25% of the total volume of the original reaction mixture; the amount of 0.1 n HCl solution is added to the reaction mixture, 25% of the total volume of the original reaction mixture; and stage distillation is carried out at reduced pressure, where the temperature outside the reaction vessel is less than about 45°C, and the temperature of the reaction mixture is greater than about 0°C.

In the following variant embodiment, the method includes the formation of a suspension of compound 34 in isopropylacetate.

In one variant embodiment, the suspension is heated to the boiling temperature under reflux.

In another variant embodiment, the connection 34 is in suspension for at least about 3 hours.

In some embodiments embodiment, the method is Holocene connection 34 further includes a dissolution of the connection 34 2-methyltetrahydrofuran; the addition of 0.1 n HCl to this solution to obtain a two-phase solution, which is stirred. In another variant embodiment, the method also includes separating the organic phase from the two-phase solution. In another variant embodiment, the method also includes filtering and removing solids from the organic phase. In another variant embodiment, the method also includes reducing a volume of the organic phase by approximately 50% using distillation. In another variant embodiment, the method also includes the implementation of three iterations of the following procedure: adding MeOAc, EtOAc, IPAc, t-BuOAc, tetrahydrofuran (THF), Et2O or methyl tert-butyl ether (MTBE) to the organic phase as long as the volume of the organic phase will not increase by 100%, and the decrease in the volume of the organic phase by 50% using distillation. In another variant embodiment, the method also includes adding MeOAc, EtOAc, IPAc, t-BuOAc, tetrahydrofuran (THF), Et2O or methyl tert-butyl ether (MTBE) to the organic phase as long as the volume of the organic phase will not increase by 100%. In another variant embodiment, the method also includes heating the organic phase to the boiling temperature under reflux and maintaining a specified boiling point under reflux for at least about 5 hours is. In another variant embodiment, the method also includes cooling the organic phase to a temperature in the range from about -5°to about 5°C over a period of time from 4.5 hours to 5.5 hours.

In another variant embodiment, the method of obtaining connection 34 further includes a crystallization connection 34, including the introduction of seed-rich reaction mixture containing the compound 34 in solution, using at least one crystal of essentially pure compound 34.

In another variant embodiment, the invention provides a method for obtaining compounds of formula 2

including the hydrolysis of compounds of formula 4

In the following variant embodiment, the compound of formula 4 hydrolyzing using gidrolizuemye agent in the presence of a solvent.

In some additional embodiments, the embodiment gidrolizuemye agent is a HCl, H2SO4H3PO4, Na2CO3, LiOH, KOH or NaOH.

In some embodiments embodiment the solvent used in the hydrolysis, is an H2O, methanol, ethanol, isopropanol or tert-butanol.

In the following variants embodiment, the invention provides a compound obtained by any of the methods described in this application.

p> In the following variant embodiment, the invention provides a pharmaceutical composition comprising the compound obtained by any of the methods described in this application.

In one aspect, the invention provides a method for obtaining compounds 27

comprising contacting connection 34

biological composition.

In one variant embodiment of this aspect, the biological composition comprises a biological organism selected from the group including fungi, bacteria and archaebacteria.

In one variant embodiment, the biological composition is a fungi. In the following variant embodiment, the fungi are unicellular fungi. In another variant embodiment, the mushrooms are multicellular fungi.

In the following variant embodiment, the mushrooms are multicellular fungi selected from the group including Absidia, Aspergillus, Beauveria, Botrytis, Cunninghamella, Cyathus, Gliocladium, Mortierella, Mucor, Phanerochaete, Stemphylium, Syncephalastrum and Verticillium.

In the following variant embodiment, the mushrooms are multicellular fungi selected from the group including Absidia pseudocylindrospora, Aspergillus alliaceus, Aspergillus ochraceus, Beauveria bassiana, Cunninghamella blakesleeana, Cunninghamella echinulata, Mortierella isabellina, Mucor plumbeus, Phanerochaete chrysosporium, Syncephalastrum racemosum and Verticillium theobromae.

In about the nom variant embodiment, fungi are unicellular fungi selected from the group including Candida, Debaryomyces, Geotrichum, Pichia, Rhodotorula, Saccharomyces, Sporobolomyces, Williopsis and Yarrowia.

In the following variant embodiment, the fungi are unicellular fungi selected from the group including Candida paripsilosis, Debaryomyces hansenii, Geotrichum candidum, Pichia methanolica, Pichia subpellicosa, Rhodotorula glutinis, Rhodotorula mucaliginosa, Saccharomyces cerevisiae, Sporobolomyces salmonicolor, Williopsis saturnis and Yarrowia lipolytica.

In another variant embodiment, the biological organism is archaebacteria. In the following variant embodiment, archaebacteria are Pyrococcus. In another variant embodiment, archaebacteria are Pyrococcus furiosus.

In another variant embodiment, the biological organism is a bacteria.

In the following variant embodiment, the bacteria is selected from the group comprising Lactobacillus, Pseudomonas, Rhodococcus and Streptomyces.

In the following variant embodiment, the bacteria is selected from the group comprising Lactobacillus reuterii, Pseudomonas methanolica, Rhodococcus erythropolis, Streptomyces griseus, Streptomyces griseolus, Streptomyces platensis and Streptomyces rimosus.

In another variant embodiment, the biological composition comprises Streptomyces rimosus or fragment.

In one variant embodiment of this aspect, the biological composition includes a solvent. In the following variant embodiment, the solvent includes water. In another variant embodiment, the solution of the tel represents the buffer. In another variant embodiment, the solvent is califofnia buffer having a pH of about 7.

In one aspect, the invention provides a method for obtaining compounds 28

including interaction connection 34

biological composition.

In one variant embodiment of this aspect, the biological composition comprises a biological organism selected from the group including fungi, bacteria and archaebacteria.

In one variant embodiment, the biological composition is a fungi. In the following variant embodiment, the fungi are unicellular fungi. In another variant embodiment, the mushrooms are multicellular fungi.

In the following variant embodiment, the mushrooms are multicellular fungi selected from the group including Absidia, Aspergillus, Beauveria, Botrytis, Cunninghatnella, Cyathus, Gliocladium, Mortierella, Mucor, Phanerochaete, Stemphylium, Syncephalastrum and Verticillium.

In the following variant embodiment, the mushrooms are multicellular fungi selected from the group including Absidia pseudocylindrospora, Aspergillus alliaceus, Aspergillus ochraceus, Beauveria bassiana, Cunninghatnella blakesleeana, Cunninghamella echinulata, Mortierella isabellina, Mucor plumbeus, Phanerochaete chrysosporium, Syncephalastrum racemosum and Verticillium theobromae.

In another variant embodiment, the fungi are unicellular fungi, select the data group, including Candida, Debaryomyces, Geotrichum, Pichia, Rhodotorula, Saccharomyces, Sporobolomyces, Williopsis and Yarrowia.

In the following variant embodiment, the fungi are unicellular fungi selected from the group including Candida paripsilosis, Debaryomyces hansenii, Geotrichum candidum, Pichia methanolica, Pichia subpellicosa, Rhodotorula glutinis, Rhodotorula mucaliginosa, Saccharomyces cerevisiae, Sporobolomyces salmonicolor, Williopsis saturnis and Yarrowia lipolytica.

In another variant embodiment, the biological organism is archaebacteria. In the following variant embodiment, archaebacteria are Pyrococcus. In another variant embodiment, archaebacteria are Pyrococcus furiosus.

In another variant embodiment, the biological organism is a bacteria.

In the following variant embodiment, the bacteria is selected from the group comprising Lactobacillus, Pseudomonas, Rhodococcus and Streptomyces.

In the following variant embodiment, the bacteria is selected from the group comprising Lactobacillus reuterii, Pseudomonas methanolica, Rhodococcus erythropolis, Streptomyces griseus, Streptomyces griseolus, Streptomyces platensis and Streptomyces rimosus.

In one variant embodiment of this aspect, the biological composition comprises Streptomyces rimosus or fragment.

In one variant embodiment of this aspect, the biological composition includes a solvent. In the following variant embodiment, the solvent includes water. In another variant embodiment, the solvent is a buffer. In another variant of the embodiment the Oia, the solvent is califofnia buffer having a pH of about 7.

III. TOTAL SYNTHESIS

The compounds of formula 1 can be synthesized according to Scheme 1.

In figure 1, anilines of formula 3, where R2, R4and R5optionally and independently substituted by functional groups, defined above, and where these functional groups optionally and independently have attached thereto a protective group, is subjected to the interaction with carbonnanotube intermediate compounds of formula 2 in the conditions of the condensation reaction. Derivatives of formula 1, which have attached to them one or more protective groups, can then be subjected to the procedure of removing protection from receiving unprotected derivatives of formula 1.

The condensation reaction shown in Scheme 1 can be carried out by dissolving used in the reaction of substances in a suitable solvent, processing the obtained solution suitable binding reagent, optionally in the presence of a suitable base.

Anilines of formula 3, where R4is a protected 1-hydroxy-2-methylpropan-2-yl, can be synthesized in accordance with Scheme 2.

Alternatively, anilines of formula 3, where R4is a protected 1-hydroxy-2-methylpropan-2-yl, which can be synthesized in accordance with Scheme 3.

Anilines of formula 3, where R4and R5together with the phenyl ring to which they are bound, form 3,3-dimethylbenzofuran-2(3H)-he can be synthesized in accordance with Scheme 4.

Alternatively, anilines of formula 3, where R4and R5together with the phenyl ring to which they are bound, form 3,3-dimethylbenzofuran-2(3H)-he can be synthesized in accordance with Scheme 5.

Anilines of formula 3, where R5is a protected hydroxyl, can be synthesized in accordance with Scheme 6.

Dihydropyrimidinase acid of formula 2 can be synthesized in accordance with Scheme 7, where the aniline derivative is subjected to the reactions of addition of conjugate to EtOCH=C(COOEt)2with subsequent thermal rearrangement and hydrolysis.

IV. APPLICATIONS AND WAYS to USE

Pharmaceutically acceptable compositions

In one aspect of the present invention, are provided pharmaceutically acceptable compositions, such compositions include any of the compounds described in this application, and optionally include pharmaceutically reception is emy media adjuvant or excipient. In some embodiments, embodiments, these compositions optionally further include one or more additional therapeutic agents.

Also it should be clear that some compounds of the present invention can exist in free form for treatment, or, if it is appropriate, in the form of a pharmaceutically acceptable derivative or prodrug. In accordance with the present invention, pharmaceutically acceptable derivative or prodrug includes, but is not limited to, pharmaceutically acceptable salts, esters, salts of such esters, or any other product or accession or derivative which upon administration to a patient in need this, is able to provide, directly or indirectly, a compound that is described in this application, or its metabolite or residue.

Used in this application, the term "pharmaceutically acceptable salt" refers to those salts which are, in accordance with suspended medical assessment, are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic reactions, etc., and are commensurate with a reasonable ratio of benefit/risk. "Pharmaceutically acceptable salt" means any non-toxic salt is whether salt of ester compounds of the present invention, which upon administration to the recipient is capable of providing, either directly or indirectly, a compound of the present invention or possessing inhibitory activity of the metabolite or residue of such compounds.

Pharmaceutically acceptable salts are well known in the prior art. For example, S. M. Berge, et al. Describe pharmaceutically acceptable salts in J. Pharmaceutical Sciences, 1977, 66, 1-19, which is incorporated into the present application by reference. Pharmaceutically acceptable salts of the compounds of the present invention include salts derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid additive salts include salts of an amino group formed with inorganic acids such as hydrochloric acid, Hydrobromic acid, phosphoric acid, sulfuric acid, Perlina acid, or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, bansilalpet, benzoate, bisulfate, borate, butyrate, comfort, camphorsulfonic is t, citrate, cyclopentanepropionate, digluconate, dodecyl sulphate, edisylate (etandisulfonat), aconsultant, formate, fumarate, glucoheptonate, glycerol, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxyethanesulfonic, lactobionate, lactate, laurate, lauryl, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluensulfonate, undecanoate, valerate and the like. Salts derived from appropriate bases include alkali metal salts, alkaline earth metals, ammonium and N+(C1-4alkyl)4. The present invention also envisions the quaternization of any basic nitrogen-containing groups of the compounds disclosed in this application. Water - or oil-soluble or dispersible products may be obtained by using such quaternization. Representative salts of alkaline or alkaline-earth metals include sodium, lithium, potassium, calcium, magnesium and the like. In addition, pharmaceutically acceptable salts include, if it is appropriate, nontoxic ammonium cations, Quaternary ammonium and amine formed using counterions such as halide, hydroxide, carboxyl is t, sulfate, phosphate, nitrate, lower alkylsulfonate and arylsulfonate.

As described above, the pharmaceutically acceptable compositions of the present invention optionally include a pharmaceutically acceptable carrier, adjuvant or excipient, which, as used in this application includes any and all solvents, diluents, or other liquid carriers, substances that contribute to the dispersion or suspensioni, surface active agents, isotonic agents, thickeners or emulsifiers, preservatives, solid binders, lubricants and the like, as is appropriate for a particular dosage form, which is desirable. Remington''s Pharmaceutical Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980) discloses various carriers used in formulating pharmaceutically acceptable compositions and known methods of obtaining them. Except only in those cases where any traditional medium used as a carrier, is incompatible with the compounds of the present invention, for example, causing any undesirable biological effect or otherwise interacting in an adverse manner with any other component(components) pharmaceutically acceptable composition, its use is envisaged as covered by the scope nastojasih the invention. Some examples of substances which can serve as pharmaceutically acceptable carriers include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid or potassium sorbate, a mixture of partial glycerides of saturated vegetable fatty acids, water, salts or electrolytes, such as preteenslut, secondary, acidic sodium phosphate, potassium phosphate, sodium chloride, zinc salts, colloidal silicon dioxide, magnesium trisilicate, polyvinylpyrrolidone, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, lanolin, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; powdered tragakant; malt; gelatin; talc; excipients such as cocoa butter and waxes for suppositories; oils such as peanut oil, cotton seed; safflower oil; sesame oil; olive oil; corn oil and soybean oil; glycols; such a propylene glycol or polyethylene glycol; esters, such as etiloleat and tillaart; agar; buffering agents such as hydroxide MAGN who I am and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; ringer's solution; ethyl alcohol and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, and colouring agents, substances promoting the release of shapes, substances, coatings, sweeteners, fragrances and flavors, preservatives and antioxidants can also be present in the compositions in accordance with the order, as it deems necessary specialist involved in formulating the composition.

Application of compounds and pharmaceutically acceptable compositions

In another aspect the present invention provides a method of treating or attenuating the severity of the condition, disease or disorder associated with CFTR mutation. In some embodiments embodiment, the present invention provides a method of treating a condition, disease or disorder associated with deficiency of CFTR activity, the method includes introducing a composition comprising a compound of formula 1, to a subject, preferably a mammal in need of it.

In another aspect, the invention also provides a method of treating or attenuating the severity of the disease in a patient, comprising the introduction of a specified patient one of the compositions defined in the present application, and the specified C is bolovanje selected from such diseases, as cystic fibrosis, asthma, caused by Smoking COPD (chronic obstructive pulmonary disease), chronic bronchitis, rhinosinusitis, constipation, pancreatitis, pancreatic insufficiency cancer, male infertility caused by congenital bilateral absence of the VAS deferens (CBAVD), uncomplicated form of pulmonary disease, idiopathic pancreatitis, allergic bronchopulmonary aspergillosis (ABPA), liver disease, hereditary emphysema, hereditary hemochromatosis, coagulation-fibrinolysis deficiencies, such as protein deficiency, hereditary angioedema type 1, lipid processing, such as familial hypercholesterolaemia, chylomicronemia type 1, abetalipoproteinemia, lysosomal storage disorders, such as disease cellular inclusions/disease Deri, mucopolysaccharidosis disease Sandhof/Tay-Sachs disease criglernajjar Najjar syndrome type II, polyendocrinopathy/hyperinsulemia, diabetes, dwarfism of Larona, myeloperoxidase deficiency, primary hypoparathyroidism, melanoma, glycans CDG (congenital diseases of glycosylation type 1, congenital hyperthyroidism, imperfect osteogenesis, hereditary hypofibrinogenemia, the lack of an activated clotting time of blood, diabetes insipidus (ND), insipidus neurotically the Diab is, neurogenic diabetes insipidus, muscular atrophy, Charcot-Marie-Toot disease Pelizaeus-Merzbacher neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, progressive supranuclear paralysis, atrophy of the Peak, some polyglutamine neurological disorders such as Huntington's disease, spinal-cerebellar ataxia type I, spinal and bulbar muscular atrophy, dentato-rubro-pallido-Lisova atrophy and myotonica dystrophy, as well as spongiform encephalopathies, such as hereditary disease Creutzfeldt-Jakob disease (due to defective processing of the prion protein), Fabry disease, syndrome Straussler-Sheinker, COPD (chronic obstructive pulmonary disease), dry eye syndrome, or sjögren's disease, osteoporosis, osteopenia, bone healing and bone growth (including the restoration of the bone, bone regeneration, reducing bone resorption and increasing bone deposition), the syndrome Goreme, chloride of kalapati, such as congenital myotonia form (Thomson and Becker), the syndrome Bartter type III, dent disease, hyperekplexia, epilepsy, hyperekplexia, lysosomal disease accumulation, Angelman syndrome, and primary ciliary dyskinesia (PCD), the term for inherited disorders of the structure and/or function resisteth structure is, including PCD with transposition of internal organs (also known as syndrome Addition, PCD without transposition of internal organs and ciliary aplasia.

In some embodiments embodiment the method comprises the treatment or mitigation of the severity of cystic fibrosis in a patient, comprising the introduction of a specified patient one of the compositions defined in the present application. In some embodiments embodiments, the patient has a mutant form of human CFTR. In other variants of the embodiment, the patient has one or more of the following mutations ΔF508, R117H, and G551D human CFTR. In one variant embodiment, the method includes the treatment or mitigation of the severity of cystic fibrosis in a patient possessing the ΔF508 mutation of human CFTR comprising the introduction of a specified patient one of the compositions defined in the present application. In one variant embodiment, the method includes the treatment or mitigation of the severity of cystic fibrosis in a patient possessing the G551D mutation of human CFTR comprising the introduction of a specified patient one of the compositions defined in the present application. In one variant embodiment, the method includes the treatment or mitigation of the severity of cystic fibrosis in a patient possessing the ΔF508 mutation of human CFTR on at least one allele, including the introduction of a specified patient one of the compositions, particularly the x in this application. In one variant embodiment, the method includes the treatment or mitigation of the severity of cystic fibrosis in a patient possessing the ΔF508 mutation of human CFTR on both alleles, including the introduction of a specified patient one of the compositions defined in the present application. In one variant embodiment, the method includes the treatment or mitigation of the severity of cystic fibrosis in a patient possessing the G551D mutation of human CFTR on at least one allele, including the introduction of a specified patient one of the compositions defined in the present application. In one variant embodiment, the method includes the treatment or mitigation of the severity of cystic fibrosis in a patient possessing the G551D mutation of human CFTR on both alleles, including the introduction of a specified patient one of the compositions defined in the present application.

In some embodiments the embodiment of the method includes the attenuation of the severity of cystic fibrosis in a patient, comprising the introduction of a specified patient one of the compositions defined in the present application. In some embodiments embodiments, the patient has a mutant form of human CFTR. In other variants of the embodiment, the patient has one or more of the following mutations ΔF508, R117H, and G551D human CFTR. In one variant embodiment, the method includes the attenuation of the severity of cystic fibrosis patients with the F508 mutation of human CFTR, includes introduction to the specified patient one of the compositions defined in the present application. In one variant embodiment, the method includes the attenuation of the severity of cystic fibrosis in a patient possessing the G551D mutation of human CFTR comprising the introduction of a specified patient one of the compositions defined in the present application. In one variant embodiment, the method includes the attenuation of the severity of cystic fibrosis in a patient possessing the ΔF508 mutation of human CFTR on at least one allele, including the introduction of a specified patient one of the compositions defined in the present application. In one variant embodiment, the method includes the attenuation of the severity of cystic fibrosis in a patient possessing the ΔF508 mutation of human CFTR on both alleles, including the introduction of a specified patient one of the compositions defined in the present application. In one variant embodiment, the method includes the attenuation of the severity of cystic fibrosis in a patient possessing the G551D mutation of human CFTR on at least one allele, including the introduction of a specified patient one of the compositions defined in the present application. In one variant embodiment, the method includes the attenuation of the severity of cystic fibrosis in a patient possessing the G551D mutation of human CFTR on both alleles, including the introduction of a specified patient one of the compositions is s, defined in this application.

In some aspects, the invention provides a method of treating or attenuating the severity of osteoporosis in a patient, comprising the introduction of a given patient, the compounds of Formula 1 or its pharmaceutically acceptable salt.

In some embodiments, embodiments a method of treating or attenuating the severity of osteoporosis in a patient includes an introduction to the specified patient, essentially amorphous compound of formula 1 or its pharmaceutically acceptable salt.

In the following variants embodiment, a method of treating or attenuating the severity of osteoporosis in a patient includes an introduction to the specified patient amorphous compound of formula 1 or its pharmaceutically acceptable salt.

In some embodiments embodiment, a method of treating or attenuating the severity of osteoporosis in a patient includes an introduction to the specified patient the pharmaceutical composition described in this application.

In some aspects, the invention provides a method of treating or attenuating the severity of osteopenia in a patient, comprising the introduction of a given patient, the compounds of formula 1 or its pharmaceutically acceptable salt.

In some embodiments, embodiments a method of treating or attenuating the severity of osteopenia in a patient includes an introduction to the specified patient, essentially amorphous compound of formula 1 or its pharmaceutical is Eski acceptable salt.

In the following variants embodiment, a method of treating or attenuating the severity of osteopenia in a patient includes an introduction to the specified patient amorphous compound of formula 1.

In some embodiments embodiment, a method of treating or attenuating the severity of osteopenia in a patient includes an introduction to the specified patient the pharmaceutical composition described in this application.

In some aspects, the invention provides a method of bone healing and/or bone repair in a patient, comprising the introduction of a given patient, the compounds of formula 1 or its pharmaceutically acceptable salt.

In some embodiments, embodiments of a method of bone healing and/or bone repair in a patient includes an introduction to the specified patient, essentially amorphous compound of formula 1 or its pharmaceutically acceptable salt.

In the following variants embodiment, the method of bone healing and/or bone repair in a patient includes an introduction to the specified patient amorphous compound of formula 1 or its pharmaceutically acceptable salt.

In some embodiments embodiment, the method of bone healing and/or bone repair in a patient includes an introduction to the specified patient the pharmaceutical composition described in this application.

In some aspects, the invention provides a method of reducing resorption to the things of tissue in a patient, including the introduction of a given patient, the compounds of formula 1 or its pharmaceutically acceptable salt.

In some embodiments, embodiment a method of reducing bone resorption in a patient includes an introduction to the specified patient, essentially amorphous compound of formula 1 or its pharmaceutically acceptable salt.

In the following variants embodiment, a method of reducing bone resorption in a patient includes an introduction to the specified patient amorphous compound of formula 1 or its pharmaceutically acceptable salt.

In some aspects, the invention provides a method of increasing bone deposits in a patient, comprising the introduction of a given patient, the compounds of formula 1 or its pharmaceutically acceptable salt.

In some embodiments, embodiments of the method of increasing bone deposits in a patient includes an introduction to the specified patient, essentially amorphous compound of formula 1 or its pharmaceutically acceptable salt.

In the following variants embodiment, a method of increasing bone deposits in a patient includes an introduction to the specified patient amorphous compound of formula 1 or its pharmaceutically acceptable salt.

In some embodiments embodiment, a method of increasing bone deposits in a patient includes the introduction of the indicated patient a pharmaceutical composition, described in this the overall application.

In some aspects, the invention provides a method of treating or attenuating the severity of COPD in a patient, comprising the introduction of a given patient, the compounds of formula 1 or its pharmaceutically acceptable salt.

In some embodiments, embodiments a method of treating or attenuating the severity of COPD in a patient includes an introduction to the specified patient, essentially amorphous compound of formula 1 or its pharmaceutically acceptable salt.

In the following variants embodiment, a method of treating or attenuating the severity of COPD in a patient includes an introduction to the specified patient amorphous compound of formula 1 or its pharmaceutically acceptable salt.

In some embodiments embodiment, a method of treating or attenuating the severity of COPD in a patient includes an introduction to the specified patient the pharmaceutical composition described in this application.

In some aspects, the invention provides a method of treating or attenuating the severity caused by Smoking COPD patients, including the introduction of a given patient, the compounds of formula 1 or its pharmaceutically acceptable salt.

In some embodiments, embodiments a method of treating or attenuating the severity of COPD caused by Smoking in a patient includes an introduction to the specified patient, essentially amorphous compound of formula 1 or its pharmaceutically acceptable salt.

In the following embodiments is oblasenia, a method of treating or attenuating the severity of COPD caused by Smoking in a patient includes an introduction to the specified patient amorphous compound of formula 1 or its pharmaceutically acceptable salt.

In some embodiments embodiment, a method of treating or attenuating the severity of COPD caused by Smoking in a patient includes an introduction to the specified patient the pharmaceutical composition described in this application.

In some aspects, the invention provides a method of treating or attenuating the severity of chronic bronchitis in a patient, comprising the introduction of a given patient, the compounds of formula 1 or its pharmaceutically acceptable salt.

In some embodiments, embodiments a method of treating or attenuating the severity of chronic bronchitis in a patient includes an introduction to the specified patient, essentially amorphous compound of formula 1 or its pharmaceutically acceptable salt.

In the following variants embodiment, a method of treating or attenuating the severity of chronic bronchitis in a patient includes an introduction to the specified patient amorphous compound of formula 1 or its pharmaceutically acceptable salt.

In some embodiments embodiment, a method of treating or attenuating the severity of chronic bronchitis in a patient includes an introduction to the specified patient the pharmaceutical composition described in this application.

The fit is accordance with an alternative embodiment, the present invention provides a method of treating cystic fibrosis, comprising the stage of introduction of a given mammal an effective amount of a composition comprising the compound of the present invention.

In accordance with the present invention an "effective amount" of a compound or pharmaceutically acceptable composition is a such amount that is effective to treat or ameliorate the severity of one or more diseases, disorders or conditions mentioned above.

Another aspect of the present invention provides a method for introducing a pharmaceutical composition by oral administration to a patient at least once a day, compositions comprising the compound of formula 1. In one variant embodiment, the method includes the introduction of a pharmaceutical composition comprising a compound of formula 1, every 24 hours. In another variant embodiment, the method includes the introduction of a pharmaceutical composition comprising a compound of formula 1, every 12 hours. In the following variant embodiment, the method includes the introduction of a pharmaceutical composition comprising a compound of formula 1, three times per day. In another variant embodiment, the method includes the introduction of a pharmaceutical composition comprising a compound of formula 1, every 4 hours.

Connect the Oia and composition, in accordance with the method of the present invention, can be administered using any amount and any route of administration effective for treating or attenuating the severity of one or more of the diseases, disorders or conditions mentioned above.

In some embodiments embodiment, the compounds and compositions of the present invention are useful to treat or ameliorate the severity of cystic fibrosis in patients who exhibit residual CFTR activity in the apical membrane of respiratory tissue and nerespectarea epithelium. The presence of residual CFTR activity on the surface of the epithelium can be easily determined using methods known from the prior art, for example, the standard electrophysiological, biochemical or histochemical methods. Such methods determine CFTR activity usingin vivoorex vivoelectrophysiological methods, measurement of Cl-concentrations in the discharge sweaty or salivary glands or usingex vivobiochemical or histochemical methods for controlling the density of the cell surface. Using these methods you can easily determine the residual CFTR activity in patients heterozygous or homozygous for different mutations, including patients homozygous or heterozygous for the most is its common mutation, ΔF508.

In another variant embodiment, the compounds and compositions of the present invention are useful to treat or ameliorate the severity of cystic fibrosis in patients who have residual CFTR activity, induced or amplified using pharmacological methods or gene therapy. Such methods increase the amount of CFTR present on the cell surface inducyruya thus missing before CFTR activity in a patient or increasing the existing level of residual CFTR activity in a patient.

In one variant embodiment, the compounds and compositions of the present invention are useful to treat or ameliorate the severity of cystic fibrosis in patients within certain genotypes, demonstrating residual CFTR activity, e.g., mutations of class III (violation of regulation or gate mechanism), the mutation class IV (changing conductivity) or mutations of class V (reduced synthesis) (Lee R. Choo-Kang, Pamela L., Zeitlin, Type I, II, III, IV, and V cystic fibrosis Transmembrane Conductance Regulator Defects and Opportunities of Therapy; Current Opinion in Pulmonary Medicine 6:521 - 529, 2000). Other genotypes in patients who exhibit residual CFTR activity, include patients homozygous for one of these classes or heterozygous with any other class of mutations, including mutations of class I mutation class II or mutations that are not klassifitsirovany.

In one variant embodiment, the compounds and compositions of the present invention are useful to treat or ameliorate the severity of cystic fibrosis in patients within certain clinical phenotypes, e.g., from moderate to mild clinical phenotype that typically correlates with the amount of residual CFTR activity in the apical membrane of epithelial tissues. Such phenotypes include patients, demonstrating the insufficiency of the pancreas, or patients diagnosed with idiopathic pancreatitis and congenital bilateral absence of the VAS deferens or mild lung disease.

The exact amount needed will be different for different subjects, depending on the specific species, age and General condition of the subject, the severity of infection, specific means, method of its introduction, etc., the Compounds of the present invention are preferably formulated in dosage form containing standard dosing unit, for ease of introduction and uniform dosing. The expression "standard unit dosing", as used in this application, refers to a physically discrete unit of money that is appropriate for the patient to be treated. However, it should be clear that the total daily intake of the soya is ineni and compositions of the present invention is determined by the physician in accordance with a weighted medical assessment. The specific effective dose for any particular patient or organism will depend upon a variety of factors including the disorder to be treated, and the severity of the disorder; activity of the specific compound; the specific composition; the age, body weight, General health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound; the duration of the treatment; drugs used in combination or accidentally with the specific compound and the like factors well known in medicine. The term "patient" as used in this application, means an animal, preferably a mammal, and most preferably human.

Pharmaceutically acceptable compositions of the present invention can enter humans and other animals orally, rectally, parenterally, intracisternally, intrawaginalno, IPR, local path (for example, in the form of powders, ointments, drops or patch), buccal, in the form of oral or nasal spray or similar, depending on the severity of the infection to be treated. In some embodiments embodiment, the compounds of the present invention can be administered orally or parenterally in doses is a level from about 0.01 mg/kg to about 50 mg/kg and preferably from about 0.5 mg/kg to about 25 mg/kg of body weight of the subject per day, one or more times a day, to obtain the desired therapeutic effect.

Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents conventionally used in this field, such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, dimethylformamide, oils (in particular, oil seeds, cotton, peanut oil, corn oil, oil of seedlings seeds, olive oil, castor oil and sesame oil), glycerin, tetrahydrofurfuryl alcohol, the glycols and esters of fatty acids sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants, such as specialsee substances, emulsifiers and suspendresume agents, sweeteners, fragrances and flavorings.

Injectable preparations, for example, sterile aqueous or oily suspension for injection can be formulated according to known prior art methods for the AMI using suitable dispersing or specialsa substances and suspendida substances. A sterile preparation for injection may also be a sterile solution, suspension or emulsion for injection in a non-toxic parenterally acceptable diluent or solvent, for example, in the form of a solution in 1,3-butanediol. Acceptable fillers and solvents that can be used, you can specify the water, ringer's solution U. S. P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are traditionally used as a solvent or medium for suspension. For these purposes you can use any light non-volatile oils, including synthetic mono - or diglycerides. In addition, in preparations for injection use fatty acids such as oleic acid.

Compositions for injection can be sterilized, for example, by filtration through a retaining bacteria filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersing in sterile water or other sterile environment for injection before use.

To prolong the effect of the compounds of the present invention, it is often desirable to slow the absorption of the compound from subcutaneous or intralesional injection. This can be done using a liquid suspension of crystalline or amorphous material with poor the Odo-solubility. The absorption rate of the connection in this case depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of parenteral input form compounds obtained by dissolution or suspension of the compounds in the oil filler. Depot forms for injection is produced by the formation of matrices for microencapsulation compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of compounds with polymers and the nature of the particular polymer used, it is possible to control the rate of release of connection. Examples of other biodegradable polymers include poly(orthoevra) and poly(anhydrides). Composition depot injections also produced by the conclusion of the compounds in liposomes or microemulsions that are compatible with body tissues.

Compositions for rectal or vaginal injection preferably represent suppositories, which can be obtained by mixing the compounds of the present invention with suitable non-irritating with excipients or carriers such as cocoa butter, polyethylene glycol or wax for suppositories, which are solid at ambient temperature but is liquid at temperaturescale, and therefore melt in the rectum or vaginal cavity and release the active compound.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms the active compound is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or secondary acidic calcium phosphate and/or a) fillers or bulk substances such as starches, lactose, sucrose, glucose, mannitol and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and the Arabian gum, (c) humectants, such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate, e) slow dissolving substances such as paraffin, f) absorption accelerators such as Quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerylmonostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricating agents such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures of such substances is. In the case of capsules, tablets and pills, the dosage form may also include a buffering agent.

Solid compositions of a similar type can also be used as fillers, prisoners in soft and hard gelatin capsules using such excipients as lactose or milk sugar and high molecular weight polyethylene glycols and the like. Solid dosage forms such as tablets, pills, capsules, pills and granules can be obtained with coatings and shells, such as intersolubility coatings and other coatings well known in the field of pharmaceutical formulation. They do not necessarily contain opaque agents, and may also have such a composition that makes possible the release of the active ingredient(ingredients) only, or preferentially, in a certain part of the digestive tract, it is not necessarily slow. Examples of compositions for encapsulating substances, which can be used include polymeric substances and waxes. Solid compositions of a similar type can also be used as fillers, prisoners in soft and hard gelatin capsules using such excipients as lactose or milk sugar and high molecular weight polyethylene glycols and the like.

The active compounds also can be the in microencapsulating form with one or more excipients, as specified above. Solid dosage forms such as tablets, coated tablets, capsules, pills and granules can be obtained with coatings and shells, such as intersolubility coating that controls the release coatings and other coatings well known in the field of pharmaceutical formulation. 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 can also include, as in normal practice, additional substances other than inert diluents, for example, the lubricant for tableting and other excipients for tableting, such as magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, such dosage forms can also include buffering agents. They do not necessarily contain opaque agents, and may also have such a composition that makes possible the release of the active ingredient(ingredients) only, or preferentially, in a certain part of the digestive tract, it is not necessarily slow. Examples of compositions for encapsulating substances, which can be used include polymeric substances and waxes.

Drug formula of local administration or by percutaneous connection of the present invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalations or patches. The active ingredient is mixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers, which may be involved. Eye medications, ear drops and eye drops are also provided as covered by the scope of the present invention. In addition, the present invention provides for the use of transdermal patches, which have the added advantage of providing controlled delivery of compounds into the body. Such dosage forms are obtained by dissolving or dispersing the compound in a suitable medium. You can also use amplifiers absorption, used to enhance the penetration of compounds through the skin. Speed can be controlled either by software controlling the speed of the membrane or by dispersing the compound in a polymer matrix or gel.

Analysis of the activity of the compounds used in the present invention as a modulator of CFTR can be carried out in accordance with methods described in General in the prior art and in the Examples presented in this application.

Also it should be clear that the compounds and pharmaceutically acceptable compositions for nastasemarian can be used in combination therapy, that is, the compounds and pharmaceutically acceptable compositions can be administered simultaneously with introduction, before or after administration of one or more other desired therapeutics or medical procedures. Specific combined therapy (therapeutic agent or procedure) for use in a combined regimen should take into account compatibility of the desired therapeutics and/or procedures and the desired therapeutic effect, the achievement you want. Also it should be clear that your therapy can achieve desired effect for the same disorder (for example, the compound of the present invention can be administered simultaneously with another agent used to treat the same disorder), or they can be designed to achieve different effects (e.g., control of any adverse effects). As used in this application, additional therapeutic agents that are normally administered to treat or prevent a specific disease or condition, known as "suitable for the disease or condition to be treated."

In one variant embodiment, the additional agent selected from a mucolytic agents, bronhodilatator, antibiotic, anti-infective funds protivovospalitel the th means, the CFTR modulator other than the compounds of the present invention, or nutrients.

In one variant embodiment, the additional agent is an antibiotic. Examples of antibiotics that are useful in the present invention include tobramycin, including tobramycin powder for inhalation (TIP), azithromycin, aztreonam, including aerosol form aztreonam, amikacin, including liposomal composition, ciprofloxacin, including its composition suitable for administration by inhalation, levofloxacin, including aerosol composition, and combinations of two antibiotics, for example, fosfomicin and tobramycin.

In another variant embodiment, the additional agent is a mucolytic agent. Examples mucolytic tools that are useful in the present invention include Pulmozyme®.

In another variant embodiment, the additional agent is a bronchodilator. An example of bronchodilators include albuterol, metaproterenol sulfate, pirbuterol acetate, salmeterol or tetralin sulfate.

In another variant embodiment, an additional means is effective to restore the surface of the liquid in the Airways in the lungs. Such tools improve the movement of salt in the cells and from the cells, making the mucus in the Airways in the lungs bleekerianus, so they are more easily excreted. Examples of such tools include hypertonic saline, denufosol tetranitro ([[(3S,5R)-5-(4-amino-2-oxopyrimidine-1-yl)-3-hydroxyanisole-2-yl]methoxyhydroquinone][[[(2R,3S,4R,5R)-5-(2,4-dioxopyrimidine-1-yl)-3,4-dihydroquinoxaline-2-yl]methoxyhydroquinone]occipitotemporal]phosphate) or bronchitol (drug for inhalation based on mannitol).

In another variant embodiment, the additional agent is an anti-inflammatory agent, i.e. a tool that can reduce inflammation in the lungs. Examples of such tools that are useful in the present invention include ibuprofen, docosahexaenoyl acid (DHA), sildenafil, glutathione for inhalation, pioglitazone, hydroxychloroquine or simvastatin.

In another variant embodiment, the additional agent is a CFTR modulator other than compound 1, i.e., a tool that has the effect of modulating CFTR activity. Examples of such funds include ataluren ("PTC 124®"; 3-[5-(2-forfinal)-1,2,4-oxadiazol-3-yl]benzoic acid), sinapultide, lancemate, depeleted (human recombinant inhibitor of neutrophil elastase), cobiprostone (7-{(2R,4aR,5R,7aR)-2-[(3S)-1,1-debtor-3-methylpentyl]-2-hydroxy-6-accountpaydayloan[b]Piran-5-yl}heptane acid) or(3-(6-(1-(2,2-debtorrent[d][1,3]dioxol-5-yl)cyclop is percarboxylic)-3-methylpyridin-2-yl)benzoic acid. In another variant embodiment, the additional agent is a(3-(6-(1-(2,2-debtorrent[d][1,3]dioxol-5-yl)cyclopropanecarboxamide)-3-methylpyridin-2-yl)benzoic acid.

In another variant embodiment, the additional agent is a nutrient. Examples of such substances include pancrelipase (Deputy pancreatic enzymes), including Pancrease®, Pancreacarb®, Ultrase® or Creon®, Liprotomase® (formerly Trizytek®), Aquadeks®, or glutathione for inhalation. In one variant embodiment, additional nutrient is pancrelipase.

The amount of additional therapeutic agent present in the compositions of the present invention, should not exceed the amount, which is normally possible to introduce into the composition comprising therapeutic agent as the only active substance. Preferably, the amount of additional therapeutic agent in the compositions disclosed in the present invention must be in the range of from about 50% to 100% of the amount, which is normally possible to introduce into the composition comprising therapeutic agent as the only active substance.

Compounds of the present invention or containing pharmaceutically acceptable compositions also may be incorporated into compositions for coating an implantable medical device, such as prostheses, artificial valves, vascular grafts, stents and catheters. Accordingly, the present invention, in another aspect, includes a composition for coating an implantable device comprising the compound of the present invention described generally above and in classes and subclasses described in this application, and a carrier suitable for coating of specified implanted device. In another aspect of the present invention includes an implantable device coated with a composition including the compound of the present invention described generally above and in classes and subclasses described in this application, and a carrier suitable for coating of specified implanted device. Suitable coatings and the General description of the receiving having a coating implantable devices can be found in U.S. patent No. 6099562; 5886026; and 5304121. Coverage typically represent a biocompatible polymer material, such as polymeric hydrogels, polymethylsiloxane, polycaprolactone, polyethylene glycol, polylactic acid, ethylene vinyl acetate and mixtures thereof. The coating optionally may be additionally covered with a suitable top layer of Versiliana, polysaccharides, polyethylene glycol, phospholipids or combinations thereof, to give a composition characteristics kontroliruemom the release.

Another aspect of the present invention relates to modelirovaniya CFTR activity in a biological sample or in a patient (e.g.,in vitroorin vivo), and the method comprises the administration to the patient, or contacting the specified biological sample with a compound of formula 1 or a composition comprising the specified connection. The term "biological sample", as used in this application includes, without limitation, cell cultures or extracts; biopsy material obtained from a mammal or extracts; and blood, saliva, urine, feces, semen, tears, or other body fluids or extracts.

Modulation of CFTR in a biological sample is useful for a variety of purposes that are known to specialists in this field. Examples of such purposes include, but are not limited to, the study of CFTR in biological and pathological phenomena; and the comparative evaluation of new modulators of CFTR.

In the next version of the embodiment is provided a method of modulating activity of anionic channelin vitroorin vivoincluding the stage of contacting the specified channel with a compound of formula 1. In variants of the embodiment, the anion channel is a chloride channel or a bicarbonate channel. In other variants of the embodiment, the anion channel is a chloride channel.

In the accordance with an alternative embodiment, the present invention provides a method of increasing the number of functional CFTR in the cell membrane, including the state of engagement of the specified cell with the compound of the formula 1.

In accordance with another variant embodiment, the activity of CFTR is determined by measuring transmembrane potential. Means for measuring the potential across the membrane in a biological sample can include any of the methods known from the prior art, such as optical analysis of membrane potential or other electrophysiological methods.

Optical analysis of the membrane potential involves the use of a potential-sensitive FRET sensors described by Gonzalez and Tsien (see Gonzalez, J. E. and R. Y. Tsien (1995) "Voltage sensing by fluorescence resonance energy transfer in single cells." Biophys J 69(4): 1272-80 ; Gonzalez, J. E. and R. Y. Tsien (1997); "Improved indicators of cell membrane potential that use fluorescence resonance energy transfer" Chem Biol 4(4): 269-77) in combination with devices for measuring fluorescence changes such as the Voltage/Ion Probe Reader (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 potential-sensitive analyses based on the change of the transmission resonance energy fluorescence (FRET) between the membrane-soluble potential-sensitive dye, DiSBAC2(3) and fluorescent phospholipid, CC2-DMPE, which is attached to the outer leaf of the plasma membrane and de is there as a FRET donor. Changes in membrane potential (Vm) causes a redistribution of the negatively charged DiSBAC2(3) through the plasma membrane and the amount of transferred energy from the CC2-DMPE changes accordingly. Changes in the fluorescence emission can be monitored using VIPR™ II, which is an integrated device fluid supply and the fluorescence detector, designed for the implementation of cellular screening assays in 96 - or 384-well microtiter plates.

In one variant embodiment, the present invention provides a method of modulating CFTR activity in a biological sample, comprising the stage of contact specified biological sample with a compound of formula 1 or its pharmaceutically acceptable salt, where R1, R2, R3, R4and Y have the meanings defined above.

In one variant embodiment, the present invention provides a method of modulating CFTR activity in a biological sample, comprising the stage of contact specified biological sample with the compound obtained by the methods described in this application, having the structure:

or its pharmaceutically acceptable salt.

In one variant embodiment, the present invention provides a method of modulating CFTR in the biological sample, including the state of engagement of the specified biological sample with the compound obtained by the methods described in this application, having the structure:

or its pharmaceutically acceptable salt.

In one variant embodiment, the present invention provides a method of modulating CFTR activity in a biological sample, comprising the stage of contact specified biological sample with the compound obtained by the methods described in this application, having the structure:

or its pharmaceutically acceptable salt.

In one variant embodiment, the present invention provides a method of treating or attenuating the severity of the disease in a patient, comprising the introduction of a specified patient an effective amount of the compounds of formula 1 or its pharmaceutically acceptable salts, where R1, R2, R3, R4and Y have the meaning given above, and the disease is selected from the following: cystic fibrosis, asthma, caused by Smoking COPD (chronic obstructive pulmonary disease), chronic bronchitis, rhinosinusitis, constipation, pancreatitis, pancreatic insufficiency cancer, male infertility caused by congenital bilateral absence of the VAS deferens (CBAVD), uncomplicated form of lung for which Alemania, idiopathic pancreatitis, allergic bronchopulmonary aspergillosis (ABPA), liver disease, hereditary emphysema, hereditary hemochromatosis, coagulation-fibrinolysis deficiencies, such as protein deficiency, hereditary angioedema type 1, lipid processing, such as familial hypercholesterolaemia, chylomicronemia type 1, abetalipoproteinemia, lysosomal storage disorders, such as disease cellular inclusions/disease Deri, mucopolysaccharidosis disease Sandhof/Tay-Sachs disease criglernajjar Najjar syndrome type II, polyendocrinopathy/hyperinsulemia, diabetes, dwarfism of Larona, myeloperoxidase deficiency, primary hypoparathyroidism, melanoma, glycans CDG (congenital diseases of glycosylation type 1, congenital hyperthyroidism, imperfect osteogenesis, hereditary hypofibrinogenemia, the lack of an activated clotting time of blood, diabetes insipidus (ND), insipidus neurotically diabetes insipidus neurogenic diabetes, muscular atrophy, Charcot-Marie-Toot disease Pelizaeus-Merzbacher neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, progressive supranuclear paralysis, atrophy of the Peak, some polyglutamine neurological disorders, so it is for Huntington's disease, spinal-cerebellar ataxia type I, spinal and bulbar muscular atrophy, dentato-rubro-pallido-Lisova atrophy and myotonica dystrophy, as well as spongiform encephalopathies, such as hereditary disease Creutzfeldt-Jakob disease (due to defective processing of the prion protein), Fabry disease, syndrome Straussler-Sheinker, COPD (chronic obstructive pulmonary disease), dry eye syndrome, or sjögren's disease.

In one variant embodiment, the method includes treating or attenuating the severity of the disease in a patient by introducing a specified patient an effective amount of the compounds obtained by the methods described in this application, having the structure:

or its pharmaceutically acceptable salt.

In one variant embodiment, the method includes treating or attenuating the severity of the disease in a patient by introducing a specified patient an effective amount of the compounds obtained by the methods described in this application, having the structure:

or its pharmaceutically acceptable salt.

In another variant embodiment, the method includes treating or attenuating the severity of the disease in a patient by introducing a specified patient an effective amount of the compounds obtained by the methods described in the present for what VCE, having the structure:

or its pharmaceutically acceptable salt.

In another aspect the present invention provides a kit for use in measuring the activity of CFTR or a fragment in the biological samplein vitroorin vivoincluding (i) a composition comprising a compound of formula 1 or any of the options above embodiments; and (ii) instructions for a) contacting the composition with the biological sample and b) measuring activity of the specified CFTR or fragment.

In one variant embodiment, the kit further includes instructions for a) contacting an additional composition with the biological sample; b) measuring activity of the specified CFTR or fragment in the presence of the specified additional compound, and c) comparing the activity of the CFTR in the presence of the additional compound with the density of CFTR in the presence of a composition of formula 1.

In variants of the embodiment, the kit is used for measuring the density of CFTR.

In one variant embodiment, the kit includes a composition including the compound obtained by the methods described in this application, having the structure:

or its pharmaceutically acceptable salt.

In one variant embodiment, the kit includes a composition including the compound obtained by the methods described in this application, having the structure:

or its pharmaceutically acceptable salt.

In some embodiments embodiment the kit includes a composition including the compound obtained by the methods described in this application, having the structure:

or its pharmaceutically acceptable salt.

For a more complete disclosure of the invention described in this application, hereinafter presents the following examples. It should be clear that these examples are intended for illustrative purposes only and should not be construed as limiting in any way the present invention.

V. EXAMPLES

Getting 1: General synthesis of 4-oxo-1,4-dihydroquinoline-3-carboxylic acid (26)

The procedure to obtain ethyl 4-oxo-1,4-dihydroquinoline-3-carboxylate (25)

Compound 23 (4.77 g, while 47.7 mmol) was added dropwise to the compound 22 (10 g, and 46.3 mmol) with subsurface flow N2to displace ethanol at a temperature below 30°C for 0.5 hours. Then the solution was heated to 100-110°C and was stirred for 2.5 hours. After cooling the mixture to a temperature below 60°C was added diphenyl ether. The resulting solution was added dropwise in diphenyl ether, which was heated to 228-232°C for 1.5 hours with subsurface in the eye N 2to displace ethanol. The mixture was stirred at 228-232°C for an additional 2 hours, cooled to a temperature below 100°C and then to the precipitate of the product was added heptane. The resulting suspension was stirred at 30°C for 0.5 hours. The solids were then filtered and the precipitate washed with heptane and dried in vacuum to obtain compound 25 as a brown solid.1H NMR (DMCO-d6; 400 MHz) δ 12,25 (C), δ 8,49 (d), δ 8,10 (m), δ to 7.64 (m, δ of 7.55 (m), δ 7,34 (m), δ 4,16 (kV), δ 1,23 (t).

The procedure for obtaining 4-oxo-1,4-dihydroquinoline-3-carboxylic acid (26)

Method 1

Compound 25 (1.0 EQ.) suspended in a solution of HCl (10.0 EQ.) and H2O (11,6 about.). The suspension was heated to 85-90°C, although alternative temperature are also suitable for this hydrolysis step. For example, hydrolysis of an alternative can be performed at a temperature of from about 75 to about 100°C. In some cases, the hydrolysis is carried out at a temperature of from about 80 to about 95°C. In other cases, the stage of the hydrolysis is carried out at a temperature of from about 82 to about 93°C (for example, from about 82.5 to about 92,5°C, or from about 86 to about 89°C). After stirring at 85-90°C for approximately 6.5 hours samples were taken of the reaction mixture to determine completion of the reaction. Mixing can be carried out at any temperature suitable for the reaction is AI hydrolysis. The solution is then cooled to 20-25°C and filtered. Reactor/the precipitate was washed using H2O (2 vol. ×2). The precipitate was then washed with 2 vol. H2O before reaching a pH of 3.0. The precipitate was then dried in vacuum at 60°C to obtain compound 26.

Method 2

Compound 25 (11.3 g, 52 mmol) was added to a mixture of 10% NaOH (aqueous solution) (10 ml) and ethanol (100 ml). The solution was heated to boiling point under reflux for 16 hours, cooled to 20-25°C and then the pH was brought up to 2-3 using the 8% solution of HCl. The mixture then was stirred for 0.5 hours and filtered. The precipitate was washed with water (50 ml) and then dried in vacuum to obtain compound 26 as a brown solid.1H NMR (DMCO-d6; 400 MHz) δ 15,33 (C), δ 13,39 (C), δ 8,87 (C), δ compared to 8.26 (m), δ 7,87 (m), δ 7,80 (m), δ 7,56 (m).

Example 1: General synthesis of N-(2-tert-butyl-5-hydroxy-4-(1-hydroxy-2-methylpropan-2-yl)phenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide (27)

The General scheme of the synthesis of compound 27 is presented below, followed by the procedures of synthesis of each intermediate compounds obtained in the synthesis.

The procedure for obtaining 2-hydroxy-5-tert-butylbenzaldehyde (2)

To a stirred solution of compound 1 (700 g of 4.66 mol) in CH3CN (7.0 l) was added MgCl2(887 g to 9.32 mol), para-formald the guide (1190 g) and TEA (2.5 l, 17,9 mol) in an atmosphere of N2. The mixture was heated to boiling point under reflux for 5 hours. After cooling to room temperature, to the mixture was added 2 l of ice water, followed by addition of 6 l of 3 M HCl solution (aqueous solution). The suspension was left under stirring until then, until the solution became transparent. The organic layer was separated and the aqueous layer was extracted using MTBE (3 l ×3). The organic layers were combined and concentrated to dryness. The residue was dissolved in MTBE (4000 ml), washed with water (1000 ml ×2) and saturated saline (1000 ml), dried over anhydrous Na2SO4, was filtered, and then concentrated to obtain compound 2 as a pale yellow solid, which was used in the next reaction without further drying or purification.1H NMR (CDCl3; 400 MHz) δ 10,86 (C), δ 9,89 (C), δ to 7.59 (m), δ 7,51 (d), δ 6,94 (d), δ 10,61 (s).

The procedure for obtaining 2-(benzyloxy)-5-tert-butylbenzaldehyde (3)

To a stirred solution of compound 2 (614,5 g of 3.33 mol) in DMF (3.5 l) was added K2CO3(953 g of 6.90 mol) and benzylchloride (480 g of 3.80 mol). The mixture was heated to 90°C and kept under stirring for 3 hours. The suspension was cooled to room temperature, then was added MTBE (2 l), followed by addition of water (12 l). The mixture is then peremeshivayu for 10 minutes and the aqueous layer was separated and was extracted using MTBE (2 l ×3). The organic layers were combined and washed with water (2 l ×2) and saturated brine (1.5 l × 1) and then concentrated to obtain compound 3 as a pale yellow solid.1H NMR (DMCO-d6; 400 MHz) δ 10,42 (C), δ 7,71 (m), δ 7,51 (m), δ 7,43 (m), δ 7,35 (m), δ 7.24 to (m), δ 5,27 (C), δ 1.26 in (C).

The procedure for obtaining 2-(benzyloxy)-5-tert-butylbenzyl alcohol (4)

To a stirred suspension of compound 3 (974 g, 3.63 mol) in MeOH (4000 ml) was slowly added NaBH4(121 g, 3,20 mol) at a temperature of 0-20°C. the Solution was left under stirring at the temperature of 15°C for 3 hours and then was cooled to 0°C. was added dropwise 2N HCl (aqueous solution) (1300 ml) at a temperature below 20°C. the Solution is then filtered and evaporated to dryness and the residue was dissolved in MTBE (5 l). The solution is then washed with water (2 l ×2) and saturated brine (1.5 l ×1). Evaporation of the solvent gave compound 4 as a pale yellow solid, which was used in the next stage of the reaction without additional purification.1H NMR (DMCO-d6; 400 MHz) δ 7,40 (m), δ 7,32 (m), δ 7,17 (m), δ 6,91 (m), δ 5,09 (C), δ 5,00 (t), δ 4,56 (d), δ 1.26 in (C).

The procedure for obtaining 2-(benzyloxy)-5-tert-butylbenzylamine (5)

To a stirred solution of compound 4 (963 g of 3.56 mol) in anhydrous DHM (2000 ml) at 0°C was slowly added SOCl2 (535 g, 4.5 mol). The mixture was stirred at 20°C for 2 hours, then concentrated in vacuum to obtain compound 5 in the form of oil, which was used in the next reaction without further drying or purification.

The procedure for obtaining 2-(benzyloxy)-5-tert-butylbenzonitrile (6)

To a stirred solution of compound 5 (1045 g, 3.54 mol) in anhydrous DMF (1000 ml) was added KCN (733 g, 11.3 mol). The mixture was stirred at 35°C for 24 hours, then poured into water (10 l). Added ethyl acetate (4 l) and the mixture was stirred for 30 minutes. The organic layer was then separated and the aqueous layer was extracted with ethyl acetate (3000 ml ×2). The organic layers were combined and washed with water (4 l ×2) and saturated saline solution (3 l ×1), then concentrated in vacuum to obtain compound 6 as a yellow solid.1H NMR (DMCO-d6; 400 MHz) δ 7,51 (m), δ 7,37 (m), 7,02 (d), δ 5,17 (C), δ 3,88 (C) of 1.26 (C).

The procedure for obtaining 2-(2-(benzyloxy)-5-tert-butylphenyl)-2-methylpropionitrile (7)

To a stirred suspension of NaH (86 g of 2.15 mol, 60% in mineral oil) in DMF (1000 ml) was added dropwise a solution of compound 6 (100.0 g, 0,358 mol) in DMF (500 ml) at 20°C. After stirring for 30 minutes, was added dropwise MeI (205 g, 1.44 mol) in DMF (500 ml) at a temperature below 30°C for 2 hours. Suspensionemedicine for 1.5 hours at a temperature of 25-30°C, then was slowly added to ice (100 g) until then, until gas evolution ceased. The pH was brought to about 7 by the slow addition of 2N HCl solution. The mixture was diluted with water (4 l) and MTBE (2 l). The organic layer was separated and the aqueous layer was extracted using MTBE (500 ml ×2). The combined organic layers were washed with water and saturated saline solution, dried over Na2SO4, filtered and then concentrated in vacuum to obtain compound 7 as a white solid.1H NMR (DMCO-d6; 400 MHz) δ 7,56 (m), δ 7,40 (m), δ 7,34 (m), δ 7,10 (d), δ to 5.21 (C), δ 1,73 (C), δ 1,27 (s).

The procedure for obtaining 2-(2-(benzyloxy)-5-tert-butylphenyl)-2-methylpropanal (8)

To a stirred solution of compound 7 (20 g, 0,065 mol) in toluene (300 ml) was added dropwise DIBAH (80 ml, 1 M in toluene) at a temperature of from about -60 to -50°C. After stirring for 2 hours to the reaction mixture was added 6 N HCl (300 ml) and stirring was continued for 30 minutes. The organic layer was then separated, washed with 2 N HCl solution followed by washing with a solution of NaHCO3then with a saturated saline solution, dried over Na2SO4and concentrated in vacuum to obtain compound 8 in the form of oil. The product was used in next reaction without further purification.1H NMR (CDCl3; 400 M is C) δ being 9.61 (s), δ was 7.36 (m), δ 7,25 (m), δ 6.87 in (m), δ is 5.06 (m), δ 1,43 (C), δ is 1.33(C).

The procedure for obtaining 2-(2-(benzyloxy)-5-tert-butylphenyl)-2-methylpropan-1-ol (9)

To a stirred solution of compound 8 (of 9.21 g, being 0.030 mol) in MeOH (150 ml) was slowly added NaBH4(2.3 g, 0.061 mol) at 0°C. After the mixture was stirred at 20°C for 3 hours, was added 12 ml of 6 N HCl solution and the mixture was stirred for an additional 30 minutes. The solution is then concentrated to approximately one-fourth the original volume and extracted using EtOAc. The organic layer was separated and washed with water and saturated saline solution, dried using Na2SO4, filtered and then concentrated in vacuum to obtain compound 9 as a white solid.1H NMR (DMCO-d6; 400 MHz) δ 7,47 (m), δ 7,42 (m), δ 7,34 (m), δ 7,28 (m), δ 7,16 (m), δ 6,94 (m), δ 5,08 (C), δ 4,45 (t), δ 3,64 (d), δ 1.28 (in C), δ 1,25 (s).

The procedure for obtaining 2-(2-hydroxy-5-tert-butylphenyl)-2-methylpropan-1-ol (10)

Pd(OH)2(1 g) and compound 9 (9,26 g, being 0.030 mol) in MeOH (200 ml) was stirred in hydrogen atmosphere at a pressure of 20-30 psig (1,406-2,109 kg/cm2) for 16-18 hours. The mixture was then filtered through Celite® and the filtrate was concentrated to obtain compound 10 as a white solid.1H NMR (DMCO-d6; 400 MHz) δ 9,16 (C), δ 7,16 (d), δ 7,00 (m), δ 6,65 m), δ 4,71 (t), δ 3,62 (d), δ 1,27 (C), δ 1,22 (s).

The procedure for obtaining 1-((methylcarbamic)oxy)-2-(1-((methylcarbamic)oxy)-2-methylpropan-2-yl)-4-tert-butylbenzene (11)

To a stirred solution of compound 10 (23,2 g, 0.10 mol), DMAP (1.44 g) and DIEA (72,8 g of 0.56 mol) in anhydrous DHM (720 ml) was added dropwise methylchloroform (43,5 g, 0.46 mol) in DHM (160 ml) at 0°C. After the mixture was stirred at 20°C for 16 hours, the mixture was washed with water, 1 N HCl solution and saturated saline solution, dried using MgSO4and concentrated in vacuum. The residue was purified using column chromatography on silica gel (mixture 1:20 EtOAc:petroleum ether) to give compound 11 as a white solid.1H NMR (DMCO-d6; 400 MHz) δ 7,32 (m), δ 7,10 (d), δ 4.26 deaths (C), δ 3,84 (C), δ 3,64 (C), δ 1,31 (C), δ 1.28 (in with).

The procedure for obtaining 1-((methylcarbamic)oxy)-2-(1-((methylcarbamic)oxy)-2-methylpropan-2-yl)-4-tert-butyl-5-nitrobenzol (12)

To a stirred solution of compound (11) (32 g, 0,095 mol) in DHM (550 ml) was added dropwise 98% H2SO4(43 g, 0.43 mol) at 0°C. After stirring for 20 minutes at 0°C, 65% HNO3(16.2 g, to 0.17 mol) was added to the mixture dropwise at 0°C. the Mixture was then stirred at 1-10°C for 4 hours and then added to a mixture of ice-water (200 ml). The aqueous layer was separated and was extracted using DHM (200 ml ×3 and the combined organic layers were washed with water (aqueous solution), NaHCO3and saturated saline, then was dried using MgSO4and concentrated in vacuum. The residue was purified using column chromatography on silica gel (mixture 1:20 EtOAc:petroleum ether) to give the crude compound 12 in the form of oil.

The procedure for obtaining 2-tert-butyl-5-((methylcarbamic)oxy)-4-(1-((methylcarbamic)oxy)-2-methylpropan-2-yl)aniline (13)

Pd/C (2.6 g) and compound 12 (14 g, crude) was stirred in MeOH (420 ml) at room temperature in a hydrogen atmosphere at a pressure of 20-30 psig (1,406-2,109 kg/cm2) for 16-18 hours. Then the mixture was filtered through kieselguhr® and the filtrate was concentrated in vacuum. The residue was purified using column chromatography on silica gel (mixture of 1:10 EtOAc:petroleum ether) to give compound 13 as a gray solid.1H NMR (CDCl3; 400 MHz) δ 7,26 (C), δ 7,19 (C), δ 4.26 deaths (C), δ 3,89 (C), δ 3,74 (C), δ 1,40 (C), δ 1,35 (s).

The procedure for obtaining N-(2-tert-butyl-5-((methylcarbamic)oxy)-4-(1-((methylcarbamic)oxy)-2-methylpropan-2-yl)phenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide (14)

To a stirred solution of compound 26 (5.0 g, was 0.026 mol) in anhydrous DMF (120 ml) at 0°C was added EDCI (5.6 g, 0,029 mol), HOBT (3.8 g, 0,028 mol) and DIEA (6.6 g, 0,051 mol). After stirring for 1 hour the mixture was added dropwise rest the R compound 13 (3.0 g, 0,008 mol) in DHM (30 ml) at 0°C. the Mixture was stirred at 25°C for 72 hours and then concentrated in vacuum. The residue was dissolved in EtOAc (225 ml) and washed with water (120 ml ×1), 1N HCl solution (120 ml) and saturated saline solution, dried using Na2SO4and concentrated in vacuum. The residue was purified using column chromatography on silica gel (mixture of 1:1 EtOAc:petroleum ether) to give compound 14 as a white solid.1H NMR (400 MHz, CDCl3) δ 12,34 (s, 1H), 11,58 (s, 1H), 9,07 (s, 1H), 8,42 (d, 1H), 7,66 (s, 1H), 7,51 (s, 1H), 7,47 (s, 1H), 7,39 (s, 1H), 6,72 (s, 1H), 4,34 (s, 2H), 3,82 (s, 3H), 3,74 (s, 3H), of 1.41 (s, 9H), of 1.40 (s, 6H).

The procedure for obtaining N-(2-tert-butyl-5-hydroxy-4-(1-hydroxy-2-methylpropan-2-yl)phenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide (27)

To a stirred solution of KOH (1.2 g, 0.02 mol) in MeOH (80 ml) at 0°C was added compound 14 (1.9 grams, 0,0036 mol). After stirring for 2-3 hours at a temperature of 5-15°C and the mixture was concentrated to dryness. The residue is then triturated in water (10 ml), was filtered, washed using DHM and dried in vacuum for 24 hours to obtain compound 27 as a white solid.1H NMR (DMCO-d6; 400 MHz) δ 12,77 (C), δ 8,86 (C), δ 8,20 (d), δ 7,55 (d), δ 7,42 (t), δ 7,16 (kV), δ 7,02 (C), δ 6,85 (m), δ 3,55 (C), δ 1.55V (C), δ 1,35 (C), δ 1,27 (C). MS found (M+H) 409,2.

Example 2: an Alternative General synthesis of N-(2-tert-butyl-5-guide is hydroxy-4-(1-hydroxy-2-methylpropan-2-yl)phenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide (27)

The procedure to obtain methyl 2-(5-tert-butyl-2-hydroxy-4-nitrophenyl)-2-methylpropanoate (38):

A mixture of 2-bromo-4-tert-butyl-5-NITROPHENOL (15,00 g, 54,72 mmol), bis(tri-tert-butylphosphine)palladium(0) (1,422 g 2,783 mmol), zinc fluoride (2,82 g, 27,27 mmol), (MTDA) (19,35 g, 111,0 mmol) and dimethylformamide (150 ml) was heated at 70°C for 18 hours. The mixture was cooled to room temperature and diluted with water. After stirring for one hour, the aqueous phase was extracted using MTBE. The organic layer was dried in vacuum to obtain the crude product as a brown solid. Purification of the product was carried out by grinding the powder in n-heptane.1H NMR (400 MHZ, DMCO-d6) δ 10,38 (s, 1H); 7,37 (s, 1H); 6,79 (s, 1H); of 3.54 (s, 3H); 1,45 (s, 6H); 1.32 to (s, 9H).

The procedure for obtaining 4-tert-butyl-2-(1-hydroxy-2-methylpropan-2-yl)-5-NITROPHENOL (39):

1M solution of lithium aluminum hydride in THF (11,80 ml, RS 11.80 mmol) was added to a solution of methyl 2-(5-tert-butyl-2-hydroxy-4-nitrophenyl)-2-methylpropanoate (are 5.36 g, 18,15 mmol) in THF (50 ml). The mixture was stirred at ambient temperature for 3 hours and then was diluted with methanol. The mixture was acidified using 1N HCl solution (pH 1-2) and the aqueous phase extrage is ovale using MTBE. The organic phase was dried in vacuum to obtain 4-tert-butyl-2-(1-hydroxy-2-methylpropan-2-yl)-5-NITROPHENOL, which was used in the next stage without additional purification.1H-NMR (400 MHZ, DMCO-d6) δ 10,12 (s, 1H); 7,37 (s, 1H); to 6.80 (s, 1H); of 4.77 (s, 1H); 3,69-the 3.65 (m, 2H); of 1.30 (s, 9H); of 1.29 (s, 6H).

The procedure for obtaining 4-tert-butyl-2-(2-methoxycarbonylamino-1,1-dimethyl-ethyl)-5-nitro-phenyl] methylcarbamate (12)

To a solution of 4-tert-butyl-2-(1-hydroxy-2-methylpropan-2-yl)-5-NITROPHENOL (1.92 g, 7,18 mmol), triethylamine (1,745 g, 17,24 mmol) and dimethylaminopyridine (87,74 mg, 0,718 mmol) in dichloromethane (30 ml) at 0°C was slowly loaded methylchloroform (2,376 g, 25,14 mmol), keeping the temperature below 5°C. After addition the mixture was left to warm to ambient temperature and stirred until until HPLC showed complete conversion of starting material (2-8 hours). The reaction mixture was diluted with water and acidified using 1N HCl solution (pH 1-2). The aqueous phase was extracted using DHM and the combined organic layers was dried in vacuum. Raw amber semi-solid substance was recrystallized from a mixture of methanol and dichloromethane to obtain specified in the title compound as a yellow crystalline solid.1H-NMR (400 MHZ, DMCO-d6) δ to 7.67 (s, 1H); 7,52 (s, 1H); 4,30 (s, 2H); 3,86 (s, 3H); to 3.64 (s, 3H); to 1.35 (s, 9H; of 1.35 (s, 6H).

The procedure for obtaining 5-amino-4-tert-butyl-2-(2-methoxycarbonylamino-1,1-dimethyl-ethyl) - phenyl] methylcarbamate (13):

A mixture of [4-tert-butyl-2-(2-methoxycarbonylamino-1,1-dimethyl-ethyl)-5-nitro-phenyl] methylcarbamate (1.27 g, 3,313 mmol) and Pd/C (75 mg, 0.035 mmol) in methanol (50 ml) was purged with nitrogen. After purging the flask with hydrogen, the mixture was first made for 18 hours at a temperature and ambient pressure. The solution was filtered through Celite® and was dried in vacuum to obtain the product as a solid.1H-NMR (400 MHZ, DMCO-d6) δ of 6.99 (s, 1H); to 6.39 (s, 1H); 4.92 in(s, 2H); 4,13 (s, 2H); is 3.82 (s, 3H); the 3.65 (s, 3H); 1.32 to (s, 9H); of 1.23 (s, 6H).

The procedure for obtaining N-(2-tert-butyl-5-hydroxy-4-(1-hydroxy-2-methylpropan-2-yl)phenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide (27):

In a mixture of [5-amino-4-tert-butyl-2-(2-methoxycarbonylamino-1,1-dimethyl-ethyl) - phenyl] methylcarbamate (103 mg, 0.29 mmol), 4-oxo-1,4-dihydroquinoline-3-carboxylic acid (50 mg, 0.26 mmol) and pyridine (42 mg, of 0.53 mmol) in 2-Methf (3.0 ml) was loaded T3P in the form of a 50 wt.% solution 2-Methf (286 mg, 0.45 mmol). The mixture was heated to 50°C for 18 hours. After cooling to ambient temperature the mixture was diluted with water. The organic phase was separated and again washed with water. The organic phase was loaded sodium methoxide (39 mg, to 0.72 mmol) and the solution was stirred during the course is 2 hours. The reaction was suppressed by using 1 N HCl solution and after phase separation the organic phase is washed using a 0.1 N HCl solution. The organic phase is then dried in vacuum to obtain compound 27 in the form of solids. Data1H-NMR spectrum correspond to the above.

Example 3: General synthesis of 2-(5-tert-butyl-2-hydroxy-4-(4-oxo-1,4-dihydroquinoline-3-carboxamido)phenyl)-2-methylpropanoic acid (28):

The procedure for obtaining 2-(5-tert-butyl-2-hydroxyphenyl)-2-methylpropionitrile (15)

Pd (OH)2/C (2.0 g) and compound 7 (20,0 g, 0.104 g mol) was stirred in MeOH (150 ml) at room temperature in a hydrogen atmosphere at a pressure of 10 psig(0,703 kg/cm2) for 16-18 hours. The mixture is then filtered through a layer of Celite® and the filtrate was concentrated to obtain compound 15, which was used in the next stage of the reaction without additional purification.1H NMR (DMCO-d6; 400 MHz) δ 9,83 (C), δ 7.24 to (C), δ 7.18 in (m), δ to 6.80 (m), δ 1,71 (C), δ 1,24 (s).

The procedure for obtaining 4-tert-butyl-2-(2-cyanoprop-2-yl)phenylmercuriborate (16)

Stir in mixture of compound 15 (126,6 g, 0,564 mol), DMAP (6.0 g) and DIEA (188 g of 1.46 mol) in anhydrous DHM (1500 ml) at 0°C for 2 hours was added dropwise methylchloroform (110 g at 1.17 mol) in anhydrous DHM (300 ml). After stirring the 12 hours at 0°C was added a mixture of ice-water (1.5 l) and the mixture was stirred at 0°C for 30 minutes. The organic layer was separated and washed with 1N HCl solution, water and saturated salt solution. The solution DHM was dried over MgSO4and concentrated in vacuum to obtain compound 16 as a yellow solid.1H NMR (DMCO-d6; 400 MHz) δ 7,47 (m), δ 7,39 (d), δ 7.24 to (d), δ 3,84 (C), δ 1,71 (C), δ 1.30 on (with).

The procedure for obtaining 2-(1-amino-2-methyl-1-oxoprop-2-yl)-4-tert-butyl-5-nitrophenylacetate (17)

Stir in mixture of compound 16 (10.0 g, 36,3 mmol) and KNO3(5,51 g of 54.5 mmol) in DHM (1000 ml) was added dropwise 98% solution of H2SO4(145,4 g of 1.45 mol) at 0°C. the Mixture was stirred at 30°C for 4 days. Layer H2SO4then was separated and poured into a mixture of ice water (50 g) and then was extracted using DHM (100 ml ×3). The combined organic layers were washed with water, aqueous solution of NaHCO3and saturated saline, then was dried over MgSO4and concentrated in vacuum. The residue was purified using column chromatography on silica gel (mixture of petroleum ether/EtOAc 20:1→10:1→5:1→3:1) obtaining of compound 17 as a yellow solid.1H NMR (CDCl3; 400 MHz) δ 8,05 (C), δ 7,74 (C), δ to 7.61 (s), δ 7,32 (C), δ 5,32 (C), δ 3,91 (C) to 3.92 δ (), δ 1,62 (C), δ 1,59 (C), δ 1,42 (C), δ 1,38 (s).

The procedure for obtaining 2-(5-tert-butyl-2-hydroxy-4-nitrophenyl)-2-methylpropanoic acid (18)

To a mixture of compound 17 (7,3 g, 21.6 mmol) in methanol (180 ml) was added water (18 ml) and NaOH (8,64 g, 216 mmol). The solution was heated and maintained at boiling under reflux for 3 days. The solvent is evaporated in vacuum and the residue was dissolved in 140 ml of water. Then the solution was acidified to pH=2 by adding 2N HCl solution. The aqueous phase was extracted with ethyl acetate (100 ml ×3) and the combined organic phases are washed with water and saturated saline, dried over anhydrous Na2SO4and then concentrated to obtain compound 18 as a yellow solid, which was used in the next stage of the reaction without additional purification.

The procedure for obtaining 5-tert-butyl-3,3-dimethyl-6-nitrobenzofurazan-2(3H)-she (19)

To a solution of compound 18 (7,10 g of 25.2 mmol) in 710 ml of anhydrous THF was added EDCI (14.5 g, to 75.6 mmol). The resulting suspension was left under stirring at 30°C during the night. The precipitate was filtered and thoroughly washed using DHM. The filtrate was concentrated to dryness and the residue was dissolved in DHM (100 ml). The solution was washed with water (50 ml ×2) and saturated saline (50 ml ×1). Layer DHM then dried over anhydrous Na2SO4and concentrated to obtain the crude product, which was purified using column chromatography on silica gel (mixture of Peter Lany ether/EtOAc 200:1→100:1→50:1) obtaining compound 19 as a white solid. 1H NMR (CDCl3; 400 MHz) δ of 7.36 (C), δ 7,10 (C), δ 1,53 (C), δ 1.41 to (C).

The procedure to obtain 6-amino-5-tert-butyl-3,3-dimethylbenzofuran-2(3H)-it (20)

Pd/C (1.50 g) and compound 19 (3.00 g, to 1.14 mmol) suspended in THF (1500 ml) at 25°C in hydrogen atmosphere at a pressure of 30 psig (2,109 kg/cm2) for 4 hours. The mixture is then filtered through a layer of Celite® and the filtrate was concentrated in vacuum to obtain compound 20 as a white solid.1H NMR (DMCO-d6; 400 MHz) δ 7,05 (C) of 6.49 δ (), δ 5,01 (C), δ 1,35 (C), δ is 1.33 (C).

The procedure for obtaining N-(5-tert-butyl-3,3-dimethyl-2-oxo-2,3-dihydrobenzofuran-6-yl)-4-oxo-1,4-dihydroquinoline-3-carboxamide (21)

Suspension HATU (17.6 g, 46,3 mol) and compound 26 (at 8.36 g, a 44.2 mmol) in anhydrous acetonitrile (1 l) was stirred at room temperature for 1 hour. To the suspension was added compound 20 (3,40 g, 14.6 mmol) and then dropwise added DIEA (11.5g, 89,0 mmol). The mixture was stirred at 45°C for 4 days. The precipitate was filtered and thoroughly washed using DHM. The filtrate was concentrated to dryness and the residue was dissolved in DHM (200 ml) and washed using 1N HCl solution (200 ml ×2) followed by washing with 5% aqueous solution of NaHCO3(200 ml ×3) and then with saturated saline (200 ml ×1). The mixture was then dried over Na2SO4and the oxygen which has demonstrated in a vacuum. The residue was purified using column chromatography on silica gel (mixture of CH2Cl2/MeOH 100:1→50:1) to give compound 21 as a pale yellow solid.1H-NMR (400MHZ, DMCO-d6) δ 12,96 (d, J 6.4 Hz, 1H); 12,1 (s, 1H); 8,9 (d, J 6.4 Hz, 1H); with 8.33 (d, J 8 Hz, 1H); 7,84 to 7.75 (m, 2H); 7,55-of 7.48 (m, 3H); to 1.47 (s, 6H); 1,45 (s, 9H).

The procedure for obtaining 2-(5-tert-butyl-2-hydroxy-4-(4-oxo-1,4-dihydroquinoline-3-carboxamido)phenyl)-2-methylpropanoic acid (28)

To a stirred solution of compound 21 (0.9 g, 2.45 mmol) in MeOH (50 ml) at 0°C was added NaOH (1.5 g, 37.5 mmol). After stirring for 16 hours at 40°C the solvent is evaporated in vacuo, then the residue was dissolved in H2O (50 ml). The precipitate was filtered and the filtrate was washed using DHM (100 ml ×1) and ethyl acetate (100 ml ×1). The aqueous layer was acidified using 2N HCl solution until pH 1-2. The precipitate was filtered and washed using H2O (80 ml) and heptane (50 ml). It was dried in vacuum to obtain compound 28 as a white solid.1H NMR (DMCO-d6; 400 MHz) δ is 12.85 (s), δ 11,84 (C), δ 11,77 (C), δ 9,39 (C), δ 8,86 (C), δ 8,33 (C), δ 7,79 (m), δ 7,52 (m), δ 7.18 in (C), δ 7,09 (C), δ 1,44 (C), δ 1,40 (C). MS found (M+H) 423,08.

Example 4: the Second alternative synthesis of N-(2-tert-butyl-5-hydroxy-4-(1-hydroxy-2-methylpropan-2-yl)phenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide (27)

/p>

3-Necked round bottom flask with a volume of 50 ml was equipped with a magnetic stirrer, bubbling device for nitrogen and thermocouple. Into the flask was loaded connection 21 (514 mg, of 1.27 mmol) and 2-Methf (4 ml). The reaction mixture was stirred at room temperature. Added alumoweld lithium (204 mg, 6.6 mmol) in the form of solids until, until it reached 100% conversion, which was monitored using HPLC. To the reaction mixture were added salt potassium sodium 2,3-dihydroxybutanedioate tetrahydrate (50 ml 400 g/l) and MTBE (50 ml). The resulting solution was stirred for 15 minutes and then allowed to stand for 15 minutes. The organic layer was separated and the pH of the aqueous layer was brought to pH 6-7 by adding tartaric acid. The aqueous layer was extracted using MTBE. The organic layer was concentrated and dried under high vacuum to obtain specified in the connection header in the form of not-quite-white powder. Data1H-NMR spectrum was consistent with the above.

Example 5: an Alternative General synthesis of 2-(5-tert-butyl-2-hydroxy-4-(4-oxo-1,4-dihydroquinoline-3-carboxamido)phenyl)-2-methylpropanoic acid (28):

The procedure for obtaining 2-bromide carbonic acid, 4-tertbutylphenyl ester methyl ester (35)

3-Necked round bottom flask with a volume of 2 l is samali magnetic stirrer, bubbling device for nitrogen and thermocouple. Was added 2-bromo-4-tertbutylphenol (50 g, 211,7 mmol) followed by the addition DHM (1.75 l), DMAP (1.29 g, of 10.58 mmol) and Et3N (44,3 ml, 317,6 mmol). The reaction mixture was cooled to 0°C. To the reaction mixture was added dropwise methylchloroform (19,62 ml, 254 mmol). The mixture was left to warm to room temperature with stirring overnight. When the reaction was completed, the mixture was filtered through a funnel from the sintered glass. The filtrate was transferred into a separating funnel with a volume of 1 L. For damping the reaction to the filtrate was added 1N HCl (300 ml) and the organic layer was separated. The organic layer is then washed with a mixture of 291 ml of a saturated solution of NaHCO3and 100 ml of water. The layers were separated and determined that the water layer has a pH of about 8. The organic layer was concentrated and dried under high vacuum for about 16 hours to obtain specified in the connection header in the form of a clear yellow oil, which was used in the next stage of the reaction without additional purification.1H-NMR (400 MHZ, DMCO-d6) 7,66 (d, J 2.0 Hz, 1H), 7,46 (DD, J of 8.4, 2.0 Hz, 1H), 7,32 (d, J 8,4 Hz, 1H), 3,86 (s, 3H), of 1.28 (s, 9H).

The procedure for obtaining (2-bromo-4-tert-butyl-5-nitro-phenyl) methylcarbamate (36)

3-Necked round bottom flask with a volume of 2 l equipped with a magnetic stirrer, barbata is the principal device for nitrogen and thermocouple. Into the flask was loaded connection 35 (176 g, 612,9 mmol) and concentrated sulfuric acid (264 ml). The reaction mixture was cooled to -5°C-0°C. was added dropwise nitric acid (28,6 ml, 612,9 mmol) and the reaction mixture was stirred at 0°C for 2 hours. After stirring was added water (264 ml) followed by addition of MTBE (264 ml). The solution was stirred for 15 minutes, then allowed to stand for 15 minutes. The organic layer was separated, concentrated and dried under high vacuum to obtain specified in the title compound as a dark brown oil, which was used in the next stage of the reaction without additional purification.1H-NMR (400 MHZ, DMCO-d6) of 7.96 (s, 1H), 7,92 (s, 1H), with 3.89 (s, 3H), of 1.34 (s, 9H).

The procedure to obtain 2-bromo-4-tert-butyl-5-nitro-phenol (37)

(2-Bromo-4-tert-butyl-5-nitro-phenyl)methylcarbamate with 72.9 g, to 219.5 mmol) were loaded into the reactor and added DHM (291,6 ml). The yellow reaction solution was cooled, using a bath of ice. Portions was added sodium methoxide (67,04 g, 69,11 ml of a 5.4 M solution, 373,2 mmol) at a temperature of 2,2-6,9°C. After complete addition, the reaction mixture was slowly heated to ambient temperature. When the heating was completed, the reaction mixture was cooled to 0°C and extinguished using 1M HCl solution (373,2 ml, 373,2 mmol). A two-phase mixture per massively for 20 minutes and transferred into a separating funnel. The organic layer was separated and washed with water (300 ml) followed by washing with saturated saline (300 ml). The organic layer was concentrated and the crude product was dried under high vacuum. The product was further purified using supercritical fluid chromatography (SFC) on the device Berger MultiGram III (Mettler Toledo AutoChem, Newark DE). The conditions for this method were as follows: 20% methanol at a flow rate of 250 ml/min column on the PPU (30*150) from Princeton Chromatography, 100 bar, 35°C, 220 nm. Introduced in 3.5 ml 55-70 mg/ml Data were collected using the SFC utility ProNTo. The purified product obtained by SFC purification was methanol MES. Removal of the methanol was carried out by azeotropic distillation. In a round bottom flask with a volume of 1 l was downloaded dark yellow solid, 2-bromo, 4-tertbutyl, 5-nitrophenetole MES, (111,3 g to 59.9 mmol) followed by loading of heptane (500 ml). The suspension was heated to 64°C to obtain a clear solution. The solvent is kept under reduced pressure (649 mbar) for 30 minutes and then was evaporated to dryness. This procedure was repeated three times until, until he identified the absence of MeOH using1H-NMR. The product was dried under high vacuum for 16 hours to obtain the product as a dark yellow semi-solid substances.1H-NMR (400 MHZ, DMCO-d6) δ 11,2 (Shir.s, OH), 7,69 (s, 1H); 703 (C, 1H); of 1.30 (s, 9H).

The procedure for obtaining 5-tert-butyl-3,3-dimethyl-6-nitrobenzofurazan-2(3H)-she (19)

Divorcing (6,093 g, 58,92 mmol) was added in a round bottom flask after purging with nitrogen. Under the flow of nitrogen was then added Pd(tBu3P)2(2 g 3,835 mmol). Then the flask was added 2-bromo-4-tert-butyl-5-nitro-phenol (16,15 g, 58,92 mmol) dissolved in DMF (80,75 ml). The reaction mixture was a orange suspension. To the mixture was added 1-methoxy-2-methyl-prop-1-enocsi)trimethylsilane (21,61 g, 25,13 ml, 117,8 mmol) and the resulting mixture was heated up to 80°C and was stirred for 16 hours. After completion, the reaction mixture was cooled to ambient temperature and filtered through Celite®. The filtered precipitate was washed using MTBE (536,0 ml) and to the filtrate was added water (893,3 ml). The mixture was stirred for 15 minutes and advocated for an additional 15 minutes. The layers were separated and the organic phase was added a 0.5 M HCl solution (500 ml, 250,0 mmol). The layers were separated and the organic layer was washed with water (500 ml). The layers were separated and the organic layer was washed using NaCl (500 ml; 8 wt.%). The organic layer was separated and the solvent was removed in vacuum. Received the crude product as a brown crystalline solid and was then purified through a plug of silica using as eluent mesh hexane:MTBE 20:1-10:1. The fractions containing the product were combined and the solvent was removed in vacuum to obtain the pure product as a white crystalline solid.1H-NMR (400 MHZ, DMCO-d6) δ 7,80 (s, 1H); a 7.62 (s, 1H); for 1.49 (s, 6H); of 1.34 (s, 9H).

The procedure to obtain 6-amino-5-tert-butyl-3,3-dimethylbenzofuran-2(3H)-it (20)

Palladium on carbon (moisture content 5 wt.%) was placed in a round bottom flask under a stream of nitrogen. The vessel was then added 5-tert-butyl-3,3-dimethyl-6-nitro-benzofuran-2-he (4.7 g, 17,85 mmol). The vessel was then carefully loaded methanol (120 ml) under nitrogen atmosphere. The vessel was then purged with the help of N2that was pumped out the air, then filled with gaseous hydrogen. From the vessel was pumped out the air and re-filled with gaseous hydrogen, and then typed a continuous stream of gaseous hydrogen. After the introduction of gas, the reaction mixture was filtered through Celite® and the residue was washed using MeOH (300 ml). The solvent was removed in vacuum and the product was dried under high vacuum to obtain a white crystalline solid.1H-NMR (400 MHZ, DMCO-d6) δ 7,05 (s, 1H); 6.48 in (s, 1H); 5,02 (s, 2H, NH2); to 1.34 (s, 6H); of 1.30 (s, 9H).

The procedure for obtaining N-(5-tert-butyl-3,3-dimethyl-2-oxo-2,3-dihydrobenzofuran-6-yl)-4-oxo-1,4-dihydroquinoline-3-carboxamide (21)

In reaction the first vessel loaded connection 26 (2,926 g, 15,43 mmol), compound 20 (4,32 g holds 18.52 mmol), 2-Methf (35,99 ml) and then 50% T3P 2-Methf (made 13.36 g, 21.00 mmol). Added pyridine (2,441 g 2,496 ml, 30,86 mmol) and the suspension was heated at a temperature of 47.5°C±5°C for 18 hours. After heating, the reaction mixture was cooled to ambient temperature and was added 2-Methf (36) and water (30 ml). The layers were separated and the organic layer was washed with 10 wt.% citric acid solution (30 ml), water (30 ml) and twice NaHCO3(20 ml). The organic layer was washed with saturated saline (50 ml), separated and the solvent was removed in vacuum. The crude product was dissolved in MTBE (100 ml) and was added hexane (200 ml) as an anti-solvent. The solid was besieged, and the resulting suspension was stirred for two hours. The solid was collected by vacuum filtration and the precipitate was washed with hexane. The obtained product was dried in a vacuum oven at 55°C with the release of nitrogen from receiving specified in the title compound as a beige solid.1H-NMR (400 MHZ, DMCO-d6) δ 12,96 (d, J 6.4 Hz, 1H); 12,1 (s, 1H); 8,9 (d, J 6.4 Hz, 1H); with 8.33 (d, J 8gts, 1H); 7,84 to 7.75 (m, 2H); 7,55-of 7.48 (m, 3H); to 1.47 (s, 6H); 1,45 (s, 9H).

The procedure for obtaining 2-(5-tert-butyl-2-hydroxy-4-(4-oxo-1,4-dihydroquinoline-3-carboxamido)phenyl)-2-methylpropanoic acid (28)

Compound 26 (81,30 mg, 0,4288 mmol) and Compound 20 (110 mg,0,4715 mmol) were loaded into a round bottom flask. Then added 2-Methf (1 ml) followed by addition of 50% T3P 2-Methf (371,4 mg, 0,5836 mmol) and pyridine (67,84 mg, 69,37 μl, 0,8576 mmol) in 2-Methf. The suspension was heated at 47,5°C±5°C during the night. After heating, the reaction mixture was cooled to ambient temperature. Was added 2-Methf (1,014 registered ml) and water (811,2 µl). The layers were separated and the organic layer was washed with water (811,2 ml) and twice with the help of NaHCO3(2 ml). The organic layer was transferred into a round bottom flask. Added LiOH (38,6 mg, 0.9 mmol) dissolved in water (2 ml) and the reaction mixture was heated to 45°C. After heating the layers were separated and the organic layer is discarded. The aqueous layer was cooled in a bath of ice and to the solution was added hydrochloric acid (of 10.72 ml of 1.0 M solution, 10.72 mmol) up until the pH reached a value of about 3-4. The aqueous layer was extracted twice using 2-Methf (5 ml) and the organic layers were combined and washed with saturated saline (5 ml). The organic layer was separated and the solvent was removed in vacuum. The obtained solid substance was dried in a vacuum oven with the release of nitrogen at 50°C with obtaining specified in the connection header.1H-NMR (400 MHZ, DMCO-d6) δ 12,89 (d, J 6.8 Hz, 1H); 11,84 (s, 1H); 11,74 (s, 1H); 9,36 (s, 1H); 8,87-8,61 (d, J 6.4 Hz ,1H); 8.34 per-8,32 (d, J 9.1 Hz, 1H); 7,83-7,745 (m, 2H); 7,17-to 7.09 (m, 1H); 7,17 (s, 1H); to 7.09 (s, 1H); USD 1.43 (s, 6H); of 1.40 (s, 9H).

Example 6: is the overall synthesis of N-(2,4-di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide (34)

The procedure for obtaining 2,4-di-tert-butylphenylphosphine (30)

Method 1

To a solution of 2,4-di-tert-butylphenol, compound 29, (10 g of 48.5 mmol) in diethyl ether (100 ml) and triethylamine (10.1 ml, for 72.8 mmol), at 0°C was added dropwise methylchloroform (7,46 ml, 97 mmol). The mixture was then left to warm to room temperature and was stirred for additional 2 hours. Then add an additional 5 ml of triethylamine and 3.7 ml of methylchloroform and the reaction mixture was stirred over night. The reaction mixture is then filtered, the filtrate was cooled to 0°C and then added an additional 5 ml of triethylamine and 3.7 ml of methylchloroform and the reaction mixture was left to warm to room temperature and then stirred for 1 hour. At this stage the reaction was almost completed and brought to completion by filtration and then washing with water (×2), followed by washing with saturated saline solution. The solution is then concentrated to a yellow oil and purified by using column chromatography to obtain compound 30.1H NMR (400 MHz, DMCO-d6) δ to 7.35 (d, J=2.4 Hz, 1H), 7,29 (DD, J=8,4,2,4 Hz, 1H), 7,06 (d, J=8,4 Hz, 1H), 3,85 (s, 3H), of 1.30 (s, 9H), of 1.29 (s, 9H).

Method 2

Into a reaction vessel were loaded the 4-dimethylaminopyridine (DMAP, and 3.16 g of 25.7 mmol) and 2,4-di-tert-butylphenol (compound 29, to 103.5 g, 501,6 mmol) was added methylene chloride (415 g, 313 ml) and the solution was stirred to dissolve all solids. Then was added triethylamine (76 g, 751 mmol) and the solution was cooled to a temperature of 0-5°C. Then was added dropwise methylchloroform (52 g, 550,3 mmol) for 2.5-4 hours to maintain a solution temperature between 0-5°C. the Reaction mixture was then slowly heated to a temperature of 23-28°C and was stirred for 20 hours. The reaction mixture is then cooled to a temperature of 10-15°C and loaded 150 ml of water. The mixture was stirred at 15-20°C for 35-45 minutes and the aqueous layer was then separated and was extracted using 150 ml of methylene chloride. The organic layers were combined and neutralized using a 2.5% solution of HCl (aqueous solution) at a temperature of 5-20°C with a final pH of 5-6. The organic layer is then washed with water and concentrated in vacuum at a temperature below 20°C to 150 ml with obtaining connection 30 in methylene chloride.

The procedure for obtaining 5-nitro-2,4-di-tert-butylphenylphosphine (31)

Method 1

To a stirred solution of compound 30 (6,77 g, 25.6 mmol) at 0°C was added dropwise 6 ml of a mixture of sulfuric acid and nitric acid 1:1. The mixture was left to warm to room temperature and was stirred for 1 hour.The product was purified, using liquid chromatography (ISCO, 120 g, 0-7% EtOAc/hexane, 38 minutes), with about 8:1 to 10:1 mixture of regioisomers of compound 31 as a white solid.1H NMR (400 MHz, DMCO-d6) δ 7,63 (s, 1H), 7,56 (s, 1H), a 3.87 (s, 3H), of 1.36 (s, 9H), of 1.32 (s, 9H). HPLC retention time to 3.92 minutes 10-99% CH3CN, the time of the experiment 5 min; ESI-MS 310 m/z (MH)+.

Method 2

To compound 30 (100 g, 378 mmol) was added DHM (540 g, 408 ml). The mixture was stirred to dissolve all solids and then cooled to a temperature of 5 to 0°C. Then was added dropwise concentrated sulfuric acid (163 g), to maintain the initial temperature of the reaction and the mixture was stirred for 4.5 hours. Then was added dropwise nitric acid (62 g) for 2-4 hours while maintaining the original reaction temperature and then stirred at this temperature for an additional 4.5 hours. The reaction mixture was then slowly added to cold water, keeping the temperature below 5°C. Then the reaction mixture was then heated to 25°C and the aqueous layer was removed and extracted with methylene chloride. The combined organic layers were washed with water, dried using Na2SO4and concentrated to a volume of 124-155 ml) was Added hexane (48 g) and the resulting mixture was again concentrated to a volume of 124-155 Jr. Next, to the mixture was added an additional amount of hexane (160 g). mesh then stirred at a temperature of 23-27°C for 15,5 hours and then filtered. To the filtered precipitate was added hexane (115 g), the mixture was heated to the boiling temperature under reflux and was stirred for 2-2,5 hours. The mixture is then cooled to a temperature of 3-7°C, stirred for an additional 1-1 .5 hours and was filtered to obtain compound 31 as a pale yellow solid.

The procedure for obtaining 5-amino-2,4-di-tert-butylphenylphosphine (32)

2,4-Di-tert-butyl-5-nitrophenylarsonic (1.00 equiv.) were loaded into a suitable reactor gidrogenizirovanii, with subsequent loading of 5% Pd/C (2.50 wt.% dry, Johnson-Matthey Type 37). The reactor was loaded MeOH (15,0 about.) and the system was closed. The system was purged with the help of N2(gas) and then brought the pressure up to 2.0 Bar using H2(gas). The reaction was carried out at the reaction temperature 25°C±5°C. After completion of the reaction, the reaction mixture was filtered and the reactor/the precipitate was washed using MeOH (4,00 about.). The obtained filtrate person to distil under vacuum at a temperature not exceeding 50°C to 8.00 about. Was added water (2,00 about.) at a temperature of 45°C±5°C. the resulting suspension was cooled to 0°C±5°C. the Suspension was kept at 0°C±5°C for at least 1 hour and filtered. The precipitate was washed once at 0°C±5°C with a mixture of MeOH/H2O (8:2) (2.00 in.). The precipitate was dried in vacuum (-0,90 bar and -0,86 bar) when the tempo is the atur 35°C-40°C to obtain compound 32. 1H NMR (400 MHz, DMCO-4) δ 7,05 (s, 1H), to 6.39 (s, 1H), 4,80 (s, 2H), 3,82 (s, 3H), of 1.33 (s, 9H), of 1.23 (s, 9H).

After completion of the reaction the mixture was diluted from about 5 to 10 volumes of MeOH (e.g., from about 6 to about 9 volumes of MeOH, from about 7 to about 8.5 volumes of MeOH, from about 7.5 to about 8 volumes of MeOH or about 7.7 volumes MeOH), was heated to a temperature of about 35±5°C, filtered, washed and dried as described above.

Obtaining N-(2,4-di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide (34).

4-Oxo-1,4-dihydroquinoline-3-carboxylic acid, compound 26, (1.0 EQ.) and 5-amino-2,4-di-tert-butylphenylmethyl, the connection 32, (1.1 EQ.) were loaded into the reactor. Was added 2-Methf (4.0 volume, relative to the acid) and then adding a 50% solution of T3P® 2-Methf (1.7 EQ.). The vessel loaded T3P, washed with 2-Methf (0,6 vol.). Then was added pyridine (2.0 equiv.) and the resulting suspension was heated to a temperature of 47.5°C±5.0°C and kept at this temperature for 8 hours. Selected sample and tested at the completion of the reaction by HPLC. After the mixture was cooled to a temperature of 25.0°C±2,5°C. For dilution of the mixture was added 2-Methf (12,5 about.). The reaction mixture is 2 times washed with water (10.0 g.). Was added 2-Methf to bring the total volume of the reaction mixture to 40.0 about. (loaded and ~16.5 in.). To this solution was added a mixture of NaOMe/MeOH (1.7 equivalents) for the implementation of the methanolysis reaction. The reaction mixture was stirred for at least 1.0 hour and the completion of the reaction was monitored using HPLC. After completion of the reaction extinguished 1N HCl solution (10,0 about.) and washed with 0.1 N HCl solution (10,0 about.). The organic solution was carefully filtered to remove any particles and placed in the second reactor. The filtered solution was concentrated at a temperature not more than 35°C (temperature of the reactor jacket) and not less than 8,0°C (internal reaction temperature) under reduced pressure to about 20. Added CH3CN to about 40. and the solution was concentrated at a temperature not above 35°C (temperature of the reactor jacket) and not less than 8,0°C (internal temperature of the reaction) to about 20. The addition of CH3CN and the cycle of concentration was repeated 2 more times for a total of 3 add CH3CN and 4 of concentration up to about 20. After the final concentration to about 20. added 16,0 about. CH3CN followed by the addition of 4.0. H2O to achieve a final concentration of about 40. 10% H2O/CH3CN relative to the original acid. This suspension was heated to a temperature 78,0°C±5.0°C (the boiling temperature under reflux). The suspension is then stirred for a period of not less than 5 hours. The suspension was cooled to a temperature of 0.0°C±5°C at the tip is of 5 hours and filtered. The precipitate was washed at a temperature of 0.0°C±5,0°C using CH3CN (5 vol.) 4 times. The obtained solid substance (compound 34) was dried in a vacuum oven at a temperature of 50.0°C±5,0°C.1H NMR (400 MHz, DMCO-d6) δ of 12.8 (s, 1H), and 11.8 (s, 1H), and 9.2 (s, 1H), 8,9 (s, 1H), and 8.3 (s, 1H), 7,2 (s, 1H), 7,9 (t, 1H), and 7.8 (d, 1H), 7.5 (t, 1H), and 7.1 (s, 1H), and 1.4 (s, 9H), and 1.4 (s, 9H).

Alternative obtaining N-(2,4-di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide (34).

4-Oxo-1,4-dihydroquinoline-3-carboxylic acid, compound 26, (1.0 EQ.) and 5-amino-2,4-di-tert-butylphenylmethyl, the connection 32, (1.1 EQ.) were loaded into the reactor. Was added 2-Methf (4,0 about., in relation to acid) and then adding a 50% solution of T3P® 2-Methf (1.7 EQ.). The vessel loaded T3P, washed with 2-Methf (0,6 vol.). Then was added pyridine (2.0 equiv.) and the resulting suspension was heated to a temperature of 47.5°C±5.0°C and kept at this temperature for 8 hours. Selected sample and tested at the completion of the reaction by HPLC. After the mixture was cooled to a temperature of 20°C±5°C. For dilution of the mixture was added 2-Methf (12,5 about.). The reaction mixture is 2 times washed with water (10,0 about.) and 2-Methf (16.5 volume) was loaded into the reactor. To this solution was added a mixture of 30% mass/mass NaOMe/MeOH (1.7 equivalents) for the implementation of the methanolysis reaction. actionnow the mixture was stirred at a temperature of 25.0°C±5,0°C for at least 1.0 hour and the completion of the reaction was monitored using HPLC. After completion of the reaction was suppressed with the help of 1.2 N HCl/H2On (10,0 about.) and washed using a 0.1 N solution of HCl/H2On (10,0 about.). The organic solution was carefully filtered to remove any particles and placed in the second reactor.

The filtered solution was concentrated at a temperature not more than 35°C (temperature of the reactor jacket) and not less than 8,0°C (internal reaction temperature) under reduced pressure to about 20. Added CH3CN to about 40. and the solution was concentrated at a temperature not more than 35°C (temperature of the reactor jacket) and not less than 8,0°C (internal temperature of the reaction) to about 20. The addition of CH3CN and the cycle of concentration was repeated 2 more times for a total of 3 add CH3CN and 4 of concentration up to about 20. After the final concentration to about 20. added 16,0 about. CH3CN followed by the addition of 4.0. H2O to achieve a final concentration of about 40. 10% H2O/CH3CN relative to the original acid. This suspension was heated to a temperature 78,0°C±5.0°C (the boiling temperature under reflux). The suspension is then stirred for a period of not less than 5 hours. The suspension was cooled to a temperature of 20°C-25°C for 5 hours and filtered. The precipitate was washed with the help of CH3CN (5 vol.), was heated to a temperature of 20°C-25°C 4 times. The obtained solid substance (compound 34) is sewed in a vacuum furnace at a temperature of 50.0°C±5,0°C. 1H NMR (400 MHz, DMCO-d6) δ of 12.8 (s, 1H), and 11.8 (s, 1H), and 9.2 (s, 1H), 8,9 (s, 1H), and 8.3 (s, 1H), 7,2 (s, 1H), 7,9 (t, 1H), and 7.8 (d, 1H), 7.5 (t, 1H), and 7.1 (s, 1H), and 1.4 (s, 9H), and 1.4 (s, 9H).

Example 7: synthesis of N-(2-tert-butyl-5-hydroxy-4-(1-hydroxy-2-methylpropan-2-yl)phenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide (27) and 2-(5-tert-butyl-2-hydroxy-4-(4-oxo-1,4-dihydroquinoline-3-carboxamido)phenyl)-2-methylpropanoic acid (28)

Streptomyces rimosus(DSM 40260) was purchased from DSMZ as a frozen culture. This culture was used for inoculation beveled Ugarov who supported and kept at 4°C. Yeast extract-malt extract-peptone (YMP) medium containing yeast extract (4 g/l), malt extract (10 g/l) and soy flour (5 g/l), were obtained and were sterilized at 130°C for 60 minutes. Five flasks containing 1 l YMP environment, directly inoculable S. rimosus of beveled Ugarov. The culture was allowed to grow for 2-3 days at 30°C with gentle agitation at approximately 100 rpm under these conditions, observed two types of growth, or a cloudy solution or spheroidal particles that have accumulated on the bottom of the flask. It has been shown that this latter type of growth leads to higher conversions in Connection 27. Cells are then centrifuged, collected and resuspendable in two flasks containing 1 l of 0.1 M potassium phosphate buffer, pH 7.0. In a flask were added ,0 g of compound 34 in 50 ml of N,N-dimethylformamide (DMF). The reaction was carried out for 24 hours at 30ºC with gentle agitation at about 100 rpm, at this point HPLC analysis showed conversion to 7.59% Connection 27 and 1,17% Connection 28.

The contents of both flasks were pooled, centrifuged at 3500 rpm for 10 minutes and resuspendable in 500 ml of methanol. This suspension was intensively stirred for 30 minutes and then centrifuged again at 6000 rpm for 10 minutes. The organic layer was collected and the process repeated twice. The methanol extracts were concentrated in vacuum to obtain 2.50 g, of 1.57 g of 1.11 g of solids, respectively. It was shown that the solids from these extracts contain 74,78-91,96% Connection 34, 7,66-19,73% Connection 27 and 0.39-5,49% Connection 28. For the selection of the connection portion 34 of the products of the oxidation process, the solids of the first two extraction were combined, suspended in 250 ml of methanol, was intensively stirred for 1 hour and filtered under vacuum. Although Compounds 27 and 28 were enriched in the filtrate (22,09 and 6.14%, respectively), the solids still contain the Compound 27 (8,96%) and Compound 28 (0,50%).

The methanol filtrate, containing approximately 2.2 g of dissolved solids, adsorbing 4.5 g of silicon dioxide, and purified flash chromatography using a gradient of 100% dichloromethane →88:12 dichloromethane/m is tonal. The fractions containing the Compound 27 was concentrated in vacuo and further dried by freeze drying to obtain 130 mg of the compound 27 (of 98.5% purity according to HPLC). The fraction containing the crude Compound 28 was also concentrated in vacuum to obtain less than 10 mg of solid substances.

Cellular precipitate after centrifugation resuspendable in 500 ml of methanol and homogenized in a BeadBeater for detachment of cells and allocate any remaining product. The organic layer was obtained by centrifugation of the homogenized suspension at 6000 rpm for 10 minutes. It was added to the solid substance, derived from the third extraction and filtered solids-rich slurry of the first two extraction, and suspended at the boiling point under reflux overnight. The suspension is then cooled and subjected to vacuum filtration to obtain 1,99 g solids. The solid was again dissolved in 300 ml of methanol, then adsorbing approximately 5 g of silicon dioxide, and purified flash chromatography using a gradient of 100% dichloromethane →94:6 dichloromethane/methanol to obtain 820 mg of a solid product containing the Connection 34 and the Connection 27, as well as other impurities. The resulting material was again subjected to column purification using bol is e high gradient of solvent (100% DHM →a mixture of 6% MeOH/94% DHM) additional 89 mg of compound 27. Range1H-NMR was consistent with the above.

Example 8: the process of recrystallization of N-(2,4-di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide (34)

Compound 34 (1.0 EQ.) were loaded into the reactor. Was added 2-Methf (20,0 about.) with the subsequent addition of 0.1 N HCl solution (5,0 about.). Two-phase solution was mixed and separated and the upper organic phase is washed twice more amount of 0.1 N HCl solution (5,0 about.). The organic solution was carefully filtered to remove any solid particles and placed in the second reactor. The filtered solution was concentrated at a temperature not above 35°C (temperature of the reactor jacket) and not more than 8,0°C (internal reaction temperature) under reduced pressure to about 10. Added isopropylacetate (IPAc) (10 vol.) and the solution was concentrated at a temperature not above 35°C (temperature of the reactor jacket) and not more than 8,0°C (internal temperature of the reaction) to about 10. Adding IPAc and concentration was repeated 2 more times for a total of 3 add IPAc and procedure 4 concentration up to about 10. After the final concentration procedure was downloaded about 10. IPAc and the suspension was heated to the boiling temperature under reflux and maintained at this temperature for 5 hours. The suspension was cooled to 0.0°C ±5°C for 5 hours and filtered. Sediment prom is Wali using IPAc (5 vol.) once. The obtained solid substance was dried in a vacuum oven at 50,0°C±5,0°C.

Example 9: General test procedure solubility at pH of 7.4

Performance analysis in shake flask was used to determine the solubility of compounds in a pH of 7.4 buffer. To calculate the concentration of compounds in solution experiments were performed using two conditions for each connection: 300 μm in 100% DMSO and 200 μm at a pH of 7.4 phosphate buffer in the presence of 2% DMSO. Each sample was left to shake overnight, then was injected into the column HPLC-UV for the determination of the peak area using the following conditions: column Phenomenex 00A-4251-B0 - 30×2.00 mm Luna 3 μm C18(2) 100A; flow rate 0.8 ml/min; volume of injected sample 20 μl; mobile phase: standard water for HPLC with 0.1% formic acid and standard acetonitrile for HPLC with 0.1% formic acid; the peak area was determined at 254 nm. Solubility in µm was calculated using the following equation: conc.=(the peak area pH 7,4)/( peak area of 300 μm DMSO standard conditions)×300 μm, the concentration of the standard conditions. Interest peaks was determined in buffer conditions on the basis of retention time (RT) of the largest peak area 300 μm DMSO standard conditions.

VI. Analyses of the ACTIVITY

Example 10: a General procedure for analysis of activity

Assays for detecting the measuring ΔF508-CFTR-potentiating properties of connections

Optical methods for the determination of membrane potential for the analysis of ΔF508-CFTR-modulating properties of connections

In the analysis using sensitive fluorescent potential dyes to measure changes in membrane potential using the tablet reader for reading fluorescence (e.g., FLIPR III, Molecular Devices, Inc.) to obtain data showing the increased level of functional ΔF508-CFTR in cells NIH 3T3. The driving force for the answer is the creation of a gradient of chloride ions due to activation of the channel by means of a single add fluid after cells were pretreated with compounds with subsequent load potential-sensitive dye.

Identification potentiating compounds

To identify ΔF508-CFTR-potentiate tools was developed HTS analysis in the format of double add. In this HTS analysis using sensitive fluorescent potential dyes to measure changes in membrane potential on FLIPR III as the gain of the gate mechanism (conductivity) ΔF508 CFTR in ΔF508 CFTR NIH 3T3 cells with a correction for temperature. The driving force for the answer is the gradient of Cl-ions due to activation of the channel by Forskolin with a single addition of liquid using a tablet reader for reading fluorescence, that the CSOs as FLIPR III, after cells were pretreated potentiate compounds (or DMSO carrier control) followed by loading dye redistribution.

Solutions

The solution in the bath #1: (in mm) NaCl 160, KCl 4.5 Is, CaCl22, MgCl21, HEPES 10, pH to 7.4 using NaOH.

The solution in the bath without chloride: Chloride salts in Solution in the bath #1 substituted gluconate salts.

Cell culture Murine NIH3T3 fibroblasts stably expressing ΔF5O8-CFTR, used for optical measurements of membrane potential. Cells were maintained at 37°C in 5% CO2and 90% humidity in the modified Dulbecco environment, Needle, supplemented with 2 mm glutamine, 10% fetal bovine serum, ×1 NEAA, β-ME, ×1 penicillin/streptomycin, and 25 mm HEPES in 175 cm2the culture flasks. For all optical analyses, the cells were sown at a density of ~20000/well in 384-well Matrigel-coated tablets and were cultured for 2 hours at 37°C, then were cultured at 27°C for 24 hours for analysis potentiate funds. For corrective analyses, cells were cultured at 27°C or 37°C with and without a connection for 16-24 hours. Electrophysiological analyses to determine ΔF508-CFTR-modulating properties of compounds.

1. Analysis using the Ussing chamber

Experiments using the Ussing chamber was performed on polari avannah epithelial cells of the respiratory tract, expressing ΔF508-CFTR to further characterize modulators ΔF508-CFTR identified in the optical assays. Non-CF and CF epithelium of the respiratory tract was isolated from bronchial tissue were cultured as described previously (Galietta, L. J. V., Lantero, S., Gazzolo, A., Sacco, A., Romano, L., Rossi, G. A., & Zegarra-Moran, O. (1998) In Vitro Cell. Dev. Biol. 34, 478-481), and were sown on Costar® Snapwell™ filters that were pre-coated with NIH3T3-conditioned medium. Four days later, the apical medium was removed and cells were grown at the interface of the air-liquid for >14 days before using them. This led to the formation of a monolayer of fully differentiated cylindrical cells, which were ciliated, a feature that is characteristic of the epithelium of the respiratory tract. Non-CF HBE were isolated from non-Smoking subjects who did not suffer from any known lung disease. CF-HBE were identified in patients homozygous for the ΔF508-CFTR.

Enable HBE grown on Costar® Snapwell™ cell culture was placed in an Ussing chamber (Physiologic Instruments, Inc., San Diego, CA) and measured the transepithelial resistance and short circuit current in the presence of basolateral →apical gradient of Cl-(IScusing a system of fixed potential (Department of Bioengineering, University of Iowa, IA). Briefly, HBE investigated in terms of registration of fixed potential (Vhold0 mV) at 37°C. Basolateral solution contained (in mm) 145 NaCl, 0,83 K2HPO4, 3,3 KH2PO4that 1.2 MgCl2, 1,2 CaCl2, 10 Glucose, 10 HEPES (pH brought up to 7.35 using NaOH), and the apical solution contained (in mm) 145 Na, 1,2 MgCl2, 1,2 CaCl2, 10 glucose, 10 HEPES (pH brought up to 7.35 using NaOH).

Identification potentiating compounds

A typical Protocol included the use of gradient concentrations of Cl-on basolateral →apical membrane. To establish this gradient, basolateral membrane used normal solutions ringer, whereas apical NaCl was replaced with equimolar amounts of sodium gluconate (titrated to pH to 7.4 using NaOH) to obtain the high concentration gradient of Cl-through the epithelium. Forskolin (10 μm) and all test compounds were added to the apical side of the inclusions in cell culture. The efficiency of the alleged ΔF508-CFTR-potentiate means were compared with known efficiency potentiate means of genistein.

2. The patch-clamp registration

General Cl-the current in ΔF508-NIH3T3 cells was monitored using the perforated patch-clamp method, as described previously (Rae, J., Cooper, K., Gates, P., & Watsky, M. (1991) J. Neurosci. Methods 37, 15-26). Determination of potential fixation was carried out at 22°C using Axopatch 200B the patch-clamp amplifier (Axon Instruments Inc., Foser City, CA). The solution in the pipette contained (in mm) 150 N-methyl-D-glucamine (NMDG)-Cl, 2 MgCl2, 2 CaCl2, 10 EGTA, 10 HEPES, and 240 μg/ml amphotericin-B (pH brought up to 7.35 using HCl). Extracellular medium contained (in mm) 150 NMDG-Cl, 2 MgCl2, 2 CaCl2, 10 HEPES (pH brought up to 7.35 using HCl). Generating pulses, data collection and analysis was performed using a PC equipped with a Digidata 1320 AJD interface in conjunction with Clampex 8 (Axon Instruments Inc.). To activate ΔF508-CFTR, 10 μm of Forskolin and 20 μm genistein was added to the bath and the ratio of the current-voltage controlled every 30 seconds.

Identification potentiating compounds

The ability of ΔF508-CFTR-potentiate funds to increase the macroscopic ΔF508-CFTR Cl-current (IΔF508) in NIH3T3 cells stably expressing ΔF508-CFTR was also investigated using the perforated patch-clamp method. Potentiate the funds identified in the optical analyses, caused a dose-dependent increase of IΔF508with the same activity and efficiency, as observed in the optical assays. In all investigated cell potential reversion before and during application potentiate funds was around -30 mV, which represents a calculated value of ECl(-28 mV).

Cell culture

Murine NIH3T3 fibroblasts stably expressing ΔF508-CFTR was used for the registration is in the configuration of the “whole cell”. Cells were maintained at 37°C in 5% CO2and 90% humidity in the modified Dulbecco environment, Needle, supplemented with 2 mm glutamine, 10% fetal bovine serum, ×1 NEAA, β-ME, ×1 penicillin/streptomycin, and 25 mm HEPES in 175 cm2flasks for cultivation. To register in the configuration of the “whole cell”, 2500-5000 cells were sown on poly-L-lysine-coated cover glasses and were cultured for 24-48 hours at 27°C before using them for testing activity potentiate means; and incubated with or without corrective compounds at 37°C for measuring the activity of correction compounds.

3. Analyses of single channels

The portal activity of wt-CFTR and temperature-corrected ΔF508-CFTR expressed in NIH3T3 cells was observed using the patch-registrations in isolated areas of the membranes in the configuration of the “inside out”, as described previously (Dalemans, W., Barbry, P., Champigny, G., Jallat, S., Dott, K., Dreyer, D., Crystal, R. G., Pavirani, A., Lecocq, J-P., Lazdunski, M. (1991) Nature 354, 526-528), using Axopatch 200B the patch-clamp amplifier (Axon Instruments Inc.). The solution in the pipette contained (in mm): 150 NMDG, 150 aspartic acid, 5 CaCl2, 2 MgCl2and 10 HEPES (pH brought up to 7.35 using Tris base). The solution in the bath contained (in mm): 150 NMDG-Cl, 2 MgCl2, 5 EGTA, 10 TES and 14 Tris base (pH brought up to 7.35 using HCl). After excision, both wt - and ΔF508-CFTR, Aktivera the Ali by adding 1 mm Mg-ATP, 75 nm catalytic subunit of cAMP-dependent protein kinase (PKA; Promega Corp. Madison, WI) and 10 mm NaF to inhibit proteinopathies, which prevented the passage of current. The potential in the pipette maintained at 80 mV. The activity of the channels analyzed in small, isolated areas of membranes containing ≤2 active channel. The maximum number of simultaneous discoveries were determined by the number of active channels in the course of the experiment. To determine the current amplitude of the single channel data recorded from 120 sec ΔF508-CFTR activity was filtered off-line at 100 Hz and then used to construct the amplitude histogram of all pixels that are customized with many features Gauss using the Bio-Patch Analysis (Bio-Logic Comp. France). General microscopic current and the probability of disclosure (P0) was determined from 120 sec for channel activity. P0was determined using the Bio-Patch or of the dependence of P0=I/i(N), where I = mean current, i = the current amplitude of the single channel, and N = number of active channels in the patch.

Cell culture

Murine NIH3T3 fibroblasts stably expressing ΔF508-CFTR, used for patch-clamp registrations in isolated areas of the membranes. Cells were maintained at 37°C in 5% CO2and 90% humidity in the modified Dulbecco environment, Needle, supplemented by the Oh 2 mm glutamine, 10% fetal bovine serum, ×1 NEAA, β-ME, ×1 penicillin/streptomycin, and 25 mm HEPES in 175 cm2flasks for cultivation. For registration of single channels, 2500-5000 cells were sown on poly-L-lysine-coated cover glasses and were cultured for 24-48 hours at 27°C before use.

The compounds of formula 1 are useful as modulators of ATP-binding cassette transporters.

OTHER VARIANTS of the INCARNATION

All publications and patents referenced in the present disclosure, is incorporated into the present application by reference to the extent as if each such publication or patent application was specifically and individually indicated as incorporated by reference. If the meaning of the terms in any of the patents or publications, incorporated by reference, contrary to the meaning of the terms used in this disclosure, it is assumed that the values of terms in the present disclosure are crucial. In addition, the above discussion discloses and describes only illustrative variants of the embodiment of the present invention. Specialists in this field can easily deduce from this discussion and from the accompanying drawings and claims that various changes, modifications and variations, without derogating the t of the essence and scope of the present invention, as set forth in the following claims.

1. The method of obtaining the compounds of formula 1,
,
including the condensation of the carboxylic acid of formula 2

with an aniline of formula 3

in the presence of TR®, where
each R2and R4independently represents a C1-6alkyl straight or branched chain, and each C1-6alkyl straight or branched chain independently and optionally substituted-OR';
each R5represents OC(O)OR' or
R4and R5taken together form the group;
y is 0;
each R' represents a C1-4alkyl group, optionally substituted by one or more groups selected from oxo and-O-C1-4is an alkyl group.

2. The method according to p. 1, further comprising splitting the group-OC(O)OR' orwith the formation of the group-HE as Deputy R5.

3. The method according to p. 2, in which the cleavage is carried out by treatment of compounds of formula 1 alcohol solvent in the presence of NaOH, KOH or sodium methoxide.

4. The method according to p. 3, wherein the alcohol solvent is a methanol.

5. The method according to any of paragraphs.1-4, in which the condensation reaction is carried out in prisutstvie the base.

6. The method according to p. 5, in which the base is a K2CO3Et3N, NMM, pyridine or DIEA.

7. The method according to p. 1, in which the condensation reaction is carried out in the presence of a solvent.

8. The method according to p. 7, in which the solvent is a acetonitrile.

9. The method according to p. 7, in which the solvent is DMF.

10. The method according to p. 7, in which the solvent is a 2-methyltetrahydrofuran.

11. The method according to p. 1, in which the condensation reaction is carried out at a reaction temperature that is maintained within the range of about 10°C-78°C.

12. The method according to p. 11, in which the condensation reaction is carried out at a reaction temperature that is maintained within the range of about 20°C-30°C.

13. The method according to p. 11, in which the condensation reaction is carried out at a reaction temperature that is maintained within the range of about 40°C and 50°C.

14. The method according to p. 11, in which the condensation reaction is carried out at a reaction temperature that is maintained within the range of about 42°C-53°C.

15. The method according to p. 1, in which the condensation reaction is carried out with stirring for at least 2 hours.

16. The method according to p. 15, in which the condensation reaction is carried out with stirring for at least 70 hours.

17. The method according to p. 15, in which the condensation reaction is carried out with stirring for, at the very measures which, 3 days.

18. The method according to p. 1, in which R2represents tert-butyl.

19. The method according to p. 1, further comprising a stage of contacting the compounds of formula 4

with a water solution of acid to obtain the compounds of formula 2.

20. The method according to p. 1, in which the compound of formula 3 is a compound of formula 40
.

21. The method according to p. 1, in which the compound of formula 3 is a compound of formula 43
.

22. The method according to p. 21, comprising a stage of contacting the compounds of formula 44

with (MTDA)

obtaining the compounds of formula 45
.

23. The method according to p. 22, further comprising a stage of recovery of the compounds of formula 45 with obtaining the compounds of formula 43.

24. The method of obtaining the compounds of formula 2
,
including contacting the compounds of formula 4

with the aqueous acid solution, where y represents 0;

25. The method of obtaining the compounds of formula 40
,
including the stage of contacting the compounds of formula 41

with methyltrimethoxysilane the tal (MTDA)

obtaining the compounds of formula 42
,
the recovery of the compounds of formula 42 with LiAlH4obtaining the compounds of formula
,
contacting the compounds with methylchloroform with obtaining the compounds of formula

and recovering the obtained compound with obtaining the compounds of formula 40.
where each R2independently represents a C1-6alkyl straight or branched chain and
each R5independently represents-HE or OS(O)OR' and
each R' independently represents a C1-4alkyl group.

26. The method of obtaining the compounds of formula 43
,
including the stage of contacting the compound having formula 44

with (MTDA)

obtaining the compounds of formula 45
,
and recovery of the compounds of formula 45 with obtaining the compounds of formula 46,
where each R2represents a C1-6alkyl straight or branched chain.

27. The method of obtaining connection 34
,
including:
(a) interactions of the connections 26
connection 32

in the presence of TR® and pyridine, using a 2-methyltetrahydrofuran as solvent, where the reaction temperature maintained within the range of about 42°C-53°C, and where the reaction is carried out for at least 2 hours, to obtain compound 33
;
(b) treating compound 33 using NaOMe/MeOH 2-methyltetrahydrofuran.

28. The method according to p. 27, in which the reaction is carried out for at least 6 hours.

29. The method according to p. 27, further comprising the formation of a suspension of compound 34 in a mixture of acetonitrile and water.

30. The method according to p. 29, in which the ratio of acetonitrile to water is about 9:1.

31. The method according to p. 29, in which the suspension is heated to a temperature in the range of approximately 73°C to 83°C.

32. The method according to p. 29, in which the connection 34 is in suspension, at least about 3 hours.

33. The method according to p. 29, further comprising the formation of a suspension of compound 34 in isopropylacetate.

34. The method according to any of paragraphs.32 or 33, in which the suspension is heated to the boiling temperature under reflux.

35. The method according to p. 27, further including the dissolution of Compound 34 in a two-phase solution of 2-methyltetrahydrofuran and 0.1 N HCl; mixing the specified two-phase solution; the organic phase from the specified duhf the aqueous solution; filtration and removal of solids from the specified organic phase; reducing the amount specified organic phase by approximately 50%, using distillation; triple procedure: add MeOAc, EtOAc, IPAc, t-BuOAc, tetrahydrofuran (THF), Et2O or methyl tert-butyl ether (MTBE) to the organic phase as long as the volume of the organic phase will not increase by 100%, reducing the volume of the organic phase by 50%, using distillation; adding MeOAc, EtOAc, IPAc, t-BuOAc, tetrahydrofuran (THF), Et2O or methyl tert-butyl ether (MTBE) to the organic phase as long as the volume of the specified organic phase will not increase by 100%; heating the organic phase to the boiling temperature under reflux and maintaining a specified boiling point under reflux for at least about 5 hours; cooling the organic phase to a temperature in the range from about -5°C to 5°C over a period of time from about 4.5 hours to 5.5 hours.

36. The method according to p. 27, further comprising quenching the reaction mixture with 1.2 N HCl solution; thereby creating a two-phase mixture; mixing the specified two-phase mixture; the selection of the organic phase from the specified two-phase mixture; adding a 0.1 N HCl solution to the organic layer, thereby creating a two-phase mixture; mixing casanogueiros.eu mixture; the selection of the organic phase, filtering and removing solids from the specified organic phase; reducing the volume of the organic phase by approximately 50% using distillation; triple procedure: addition of acetonitrile to the organic phase as long as the volume of the specified organic phase will not increase by 100%, and the decrease in the volume of the organic phase by approximately 50%; the increase in the volume of the organic phase by approximately 100% by adding acetonitrile and then adding water, with formation of a suspension, in which the final ratio of solvent acetonitrile/water is 9:1; the heating of the specified suspension to a temperature in the range of approximately 73°C-83°C; mixing the specified suspension for at least 5 hours; and cooling the specified suspension to a temperature within about 20°C-25°C; filtering and removing solids from the specified slurry; washing the solid with acetonitrile having a temperature within about 20°C to 25°C, four times; and drying the solids under vacuum at a temperature of from about 45°C to about 55°C.

37. The method of obtaining connection 34
,
including:
(a) interactions of the connections 26

connection 32

in the presence of anhydride 2-Pro is an phosphonic acid and pyridine, using 2-methyltetrahydrofuran as solvent, where the reaction temperature maintained within the range of about 42°C-53°C, and where the reaction is carried out for at least 6 hours, to obtain compound 33
;
(b) treating compound 33 using NaOMe/MeOH 2-methyltetrahydrofuran;
(c) adding a solution of 1.2 N. HCl to the reaction mixture with the formation of the first two-phase mixture;
(d) separating the first two-phase mixture;
(e) adding a solution of 0.1 G. of HCl to the organic phase of the first two-phase mixture with the formation of the second two-phase mixture;
(f) separating the organic phase from the second two-phase mixture;
(g) reducing the amount of solvent in the organic phase of the second two-phase mixture of 50%, using distillation;
(h) increasing the volume of the organic phase of the second two-phase mixture is 100% by adding acetonitrile;
(i) performing steps (g)-(h) two additional times;
(j) adding water to the organic phase of the second two-phase mixture to obtain a suspension of compound 34 in acetonitrile and water, where the suspension is the ratio of acetonitrile to water 9:1;
(k) heating the suspension to a temperature in the range of approximately 73°C-83°C for 3 hours;
(l) cooling the suspension to a temperature in the range 20°C-25°C;
(m) filtering and separating solids from suspensions.

38. The method according to p. 7, additionally, including the dissolution of Compound 34 in a two-phase solution of 2-methyltetrahydrofuran and 0.1 N HCl; mixing the specified two-phase solution; the organic phase from the specified two-phase solution; filtering and removing solids from the specified organic phase; reducing the amount specified organic phase by approximately 50%, using distillation; triple procedure: add MeOAc, EtOAc, IPAc, t-BuOAc, tetrahydrofuran (THF), Et2O or methyl tert-butyl ether (MTBE) to the organic phase as long as the volume of the organic phase will not increase by 100%, reducing the volume of the organic phase by 50%, using distillation; adding MeOAc, EtOAc, IPAc, t-BuOAc, tetrahydrofuran (THF), Et2O or methyl tert-butyl ether (MTBE) to the organic phase as long as the volume of the specified organic phase will not increase by 100%; heating the organic phase to the boiling temperature under reflux and maintaining a specified boiling point under reflux for at least about 5 hours; cooling the organic phase to a temperature in the range from about -5°C to 5°C over a period of time from about 4.5 hours to 5.5 hours.

39. The method of obtaining connection 34
,
including:
(a) interactions of the connections 26

connection 32

in the presence of anhydride propane phosphonic acid and pyridine, using a 2-methyltetrahydrofuran as solvent, where the reaction temperature maintained within the range of about 42°C-53°C, and where the reaction is carried out for at least 2 hours, to obtain compound 33
;
(b) hydrolysis methoxycarbonyl group at phenolic oxygen atom of compound 33 by treatment of compound 33 using NaOMe/MeOH 2-methyltetrahydrofuran with the connection 34;
(c) forming a suspension of compound 34 in acetonitrile and water connection 34 in its pure form.

40. The method of obtaining the compounds of formula 8
,
including the nitration of compounds of formula 7 using a mixture of sulfuric and nitric acids in the presence of dichloromethane
,
where PG is a protective group, and each of R2and R4represents tert-butyl, and where nitrosoaniline formula 8 is purified by means of crystallization.

41. The method according to p. 40, where PG is propoxyphenyl, methanesulfonyl, 4-nitrobenzoyl, taxiformis, butoxypropyl, tert-butoxypropyl, isopropoxyphenyl or methoxypropyl.

42. The method according to p. 41, where PG is methoxypropyl.

43. FPIC is B. p. 40, additionally comprising obtaining the compounds of formula 7 by reacting the compounds of formula 6

with a reagent capable of causing adherence of the protective group to the phenolic oxygen of compounds of formula 6 in the presence of a solvent.

44. The method according to p. 43, where the solvent is a diethyl ether or methylene chloride.

45. The method according to p. 44, where the solvent represents methylene chloride.

46. The method according to p. 40, further comprising obtaining the compounds of formula 5

by restoring the compounds of formula 8.

47. The method of obtaining the compounds of formula 31, comprising contacting connection 30 with a mixture of nitric and sulfuric acids in the presence of dichloromethane
.

48. The method according to p. 47, where the reaction is quenched by adding the reaction mixture into cold water.

49. The method according to p. 47, where the water layer is extracted with dichloromethane.

50. The method according to p. 49, where dichloromethane optionally washed with water.

51. The method according to p. 47, where the reaction product is produce by crystallization in hexane.

52. The method according to p. 47, additionally comprising contacting the connection 31 in the presence of gaseous hydrogen, palladium on carbon and methanol to obtain compound 32
.



 

Same patents:

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to a method for preparing a compound of general formula VIII of enantiomeric purity min. 80% by a reaction of the compound according to general formula IV with enantiomeric pure 2-hydroxy-4-methyl-2-(trifluoromethyl)pentenoic acid to produce a compound of general formula II to be reduced to prepare a compound for general formula I to be oxidated to form an aldehyde which then reacts with an aromatic amine of formula H2N-Ar to produce a respective imine which is then reduced to prepare a compound described by formula VIII in the enantiomeric pure form. Also, it refers to methods for preparing the compound of formula I, as well as to the compounds of formula I. In general formulas

, ,

, , X1, X2, X3 is specified in fluorine, chlorine, bromine, hydroxy, methoxy, ethoxy, trifluoromethyl, amino whereas the other groups X1, X2, X3 represent a hydrogen atom.

EFFECT: preparing the non-steroid anti-inflammatory drugs.

12 cl, 5 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to novel derivatives of 1,3-dihydro-5-isobenzofurancarbonyl of formula 1, or their pharmaceutically acceptable salts, where R1 represents phenyl, phenyl substituted with C1-C6-alkyloxy, phenyl, substituted with C1-C6-alkyl, phenyl, substituted with C1-C6-dialkylamine, phenyl, substituted with halogen or thienyl; R2 is selected from group consisting of C1-C6-dialkylamino, pyrazolyl and imidazolyl, excluding C1-C6-dialkylamino, if R1 represents phenyl, phenyl substituted with halogen; n represents integer number from 1 to 3, and method of their obtaining.

EFFECT: invention also relates to pharmaceutical compositions, which include formula 1 compound, and method of treatment and prevention of premature ejaculation.

18 cl, 8 tbl, 68 ex

FIELD: chemistry.

SUBSTANCE: invention relates to pharmaceutical chemistry and specifically 2-methoxy-4-[(3aR,7aS8)-3,3,6-trimethyl-1,3,3a,4,5,7a-hexahydro-2-benzofuran-1-yl]phenol of formula 1, , having anti-Parkinson activity and can be used in medicine.

EFFECT: agent has low toxicity and can be obtained from common natural compounds 2- and 3-carenes.

5 ex, 2 tbl

FIELD: organic chemistry, chemical technology.

SUBSTANCE: invention relates to a new method for preparing phthalanes of the formula (III): wherein R2 represents halogen atom, trifluoromethyl, cyano-group or group -R-CO- wherein R represents alkyl radical comprising 1-4 carbon atoms, and its acid-additive salts. Method involves reaction of compound of salt of the general formula (II): wherein R1 represents halogen atom, and R2 has the above given values with copper cyanide.

EFFECT: simplified process, enhanced yield.

14 cl, 1 ex

FIELD: chemistry.

SUBSTANCE: invention relates to formula compound or to its pharmaceutically acceptable salt, where R represents COOH or CH2OH. Invention also relates to pharmaceutical composition based on formula I, method of modulating CFTR activity in biological sample, based on application of formula I compound, method of treatment, based on application of formula I compound, set based on formula I compound.

EFFECT: obtained are novel derivatives of quinolin-4-one, useful as CFTR modulators.

18 cl, 1 tbl, 8 ex

FIELD: chemistry.

SUBSTANCE: claimed invention relates to field of organic chemistry, namely to novel compound of formula (I), where Y and Z, each independently, are selected from group, consisting of: a) phenyl, if necessary substituted with 1 or 2 R6; b) pyridine, imidazole, thiazole, furan, triazole, quinoline or imidazopyridine, if necessary substituted with 1 R6; and c) substituent, independently selected from group, consisting of hydrogen, C1-C6alkyl or pyperidine; R1, R2 and R3, each independently selected from group, consisting of hydrogen and halogen; A and B is each independently selected from hydrogen, OH and C1-C6alkyl; RA and RB are independently selected from group, consisting of hydrogen, C1-C6alkyl and C3-C8cycloalkyl; or RA and RB together with atom, to which they are attached, form 4-6-membered heterocycle, if necessary having additionally one heteroatom or functional heterogrpoup, selected from group, consisting of -O-, -NH, -N(C1-C6-alkyl)- and -NCO(C1-C6-alkyl)-, and 6-membered heterocycle can be additionally substituted with one or two C1-C6-alkyl groups; R4 and R5, each stands for hydrogen; and each R6 is selected from Br, Cl, F, I, C1-C6-alkyl, pyrrolidine, if necessary substituted with one C1-C6-alkyl, C1-C6alkoxy, halogen-C1-C6alkyl, hydroxyl-C1-C6alkylene, -(NRARB)C1-C6alkylene and (NRARB)carbonyl; or to its individual isomer, stereoisomer or enantiomer, or their mixture, if necessary pharmaceutically acceptable salt. Invention also relates to compound of formula (II), particular compounds of formula (I) and (II), pharmaceutical composition and industrial product based on compound of formula (I) and (II), method of treating said pathological conditions, method of obtaining formula (I) compound and to intermediate compound of formula 3.

EFFECT: novel compounds, useful as inhibitors of poly(ADP-ribose)polymerase, are obtained.

50 cl, 1 tbl, 159 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to naphthalene carboxamide derivatives of general formula I which possess the properties of protein kinase or histone deacetylase inhibitors. The compounds can find application for preparing a drug for treating inflammatory diseases, autoimmune diseases, oncological disease, diseases of the nervous system and neurodegenerative diseases, allergies, asthma, cardiovascular diseases and metabolic diseases or disease related to hormonal diseases. In general formula I: , Z represents CH or N; each of the groups R1, R2 and R3 represents hydrogen, halogen, alkyl, alkoxy or trifluoromethyl; R4 represents or X represents a benzene ring or a pyridine ring; R5 represents one or more substitutes specified in a group consisting of hydrogen, halogen, alkyl, alkoxy or trifluoromethyl. The invention also refers to a method for preparing the above compounds, a pharmaceutical preparation and using them.

EFFECT: preparing the compounds which possess the properties of protein kinase or histone deacetylase inhibitors.

13 cl, 10 tbl, 6 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to new quinolone derivatives of general formula (1) or a pharmaceutically acceptable salts thereof, wherein R1 represents a hydrogen atom, a lower alkyl group, cyclo C3-8 alkyl, a lower alkyl group or a lower alkoxy, a lower alkyl group; R2 represents a hydrogen, a lower alkyl group or a halogen-substituted lower alkyl group; R3 represents a phenyl group, a difurylglyoxal group, a thienyl group or pyridyl group with each group of the above is optionally substituted by one or two groups specified in a group consisting of the following (1) to (16) in an aromatic or heterocyclic ring, presented by the above R3: (1) lower alkyl groups, (2) lower alkoxy groups, (3) halogen-substituted lower alkoxy groups; (4) a phenoxy group, (5) lower alkylthio groups, (6) a hydroxy group, (7) hydroxy lower alkyl groups, (8) halogen atoms, (9) lower alkanoyl groups, (10) lower alkoxycarbonyl groups, (11) amino groups optionally substituted by one or two lower alkyl groups, (12) carbamoyl groups optionally substituted by one or two lower alkyl groups, (13) cyclo C3-8 alkyl lower alkoxy groups, (14) pyrrolidinyl carbonyl groups, (15) morpholinyl carbonyl groups and (16) a carboxyl group; R1 represents a halogen atom; R5 represents a hydrogen atom or a halogen atom; R6 represents a hydrogen atom; and R7 represents any of the above groups (1) to (15): (1) a hydroxyl group, (2) a halogen atom, (3) a lower alkoxy group, (4) a halogen-substituted lower alkoxy group, (5) a hydroxy lower alkoxy group, (6) a lower alkoxy lower alkoxy group, (7) an amino group optionally substituted by one or two members specified in a group consisting of lower alkyl groups, lower alkoxy lower alkyl groups and cyclo C3-8 alkyl groups, (8) an amino lower alkoxy group optionally substituted in an amino group by one or two members specified in a group consisting of lower alkyl groups, lower alkanoyl group, lower alkyl sulphonyl groups and carbamoyl groups optionally substituted by one or two lower alkyl groups, (9) a cyclo C3-8 alkoxy group, (10) a cyclo C3-8 alkyl lower alkoxy group, (11) a tetrahydrofuryl lower alkoxy group, (12) a lower alkylthio group, (13) a heterocyclic group specified in a group consisting of morpholinyl groups, pyrrolidinyl groups, difurylglyoxal groups, thienyl groups and benzothienyl groups, (14) a phenyl lower alkoxy lower alkoxy group and (15) a pyrrolidinyl carbonyl group. Also, the invention refers to a pharmaceutical composition, and a preventive and/or therapeutic agent based on the compound of formula (1), using the compound of formula (1), a method of treating or preventing the above diseases, to a method of preparing the compound of formula (1).

EFFECT: there are prepared new quinolone derivatives effective for treating and/or preventing the neurodegenerative diseases, diseases caused by neurological dysfunction, or diseases induced by deterioration of mitochondrial function.

11 cl, 1 tbl, 104 ex

FIELD: chemistry.

SUBSTANCE: invention relates to derivatives with anticancer activity of formulae:

, , , , ,

R2', R3', R4', R5' and R6' are selected from H, Y(CH2)nCH3, X and (CH2)nNR8R9; Y is selected from O and S; X is selected from F, Cl and Br; R8 and R9 are selected from (CH2)nCH3; R2, R3, R4 and R5 are selected from H, Y(CH2)nCH3, X and (CH2)nNR8R9, or R3 and R4 together form -Y(CH2)nY-; R1 and R1' are selected from H, Li+, Na+, K+, N+R8R9R10R11 or benzyl, where R10 and R11 are selected from H, (CH2)nYH, (CH2)nN(CnH2n+1)(CmH2m+1) or (CH2)nCH3, where n and m are integers from 0 to 4, q is an integer from 1 to 4.

EFFECT: obtaining novel compounds with anticancer activity.

37 cl, 3 dwg, 10 ex, 2 tbl

The invention relates to the technology of known derivatives hinolincarbonova acid, in particular to a method for producing derivatives of 3-hinolincarbonova acid

The invention relates to new derivatives of 5-amino-8-methyl-7-pyrrolidineethanol-3-carboxylic acids and their stereoisomers and their pharmacologically acceptable salts, have excellent antibacterial activity, and to methods for their preparation

Method and catalyst // 2538277

FIELD: chemistry.

SUBSTANCE: invention relates to an improved method for catalytic hydrogenation of an aromatic or heteroaromatic nitro compound, substituted with one or more substitutes selected from a group consisting of halogen atoms and oxygen-containing or sulphur-containing groups, to a corresponding amine in the presence of a platinum catalyst which includes elementary platinum on a support. The platinum catalyst is modified with a molybdenum compound and a phosphorus compound, where the phosphorus compound is hypophosphorous acid or a salt thereof, or a reaction product thereof. The invention also relates to a modified supported platinum catalyst used in said method.

EFFECT: method enables selective reduction of aromatic and heteroaromatic nitro compounds, substituted with halogens or oxygen-containing or sulphur-containing substitutes, to obtain corresponding amines with high output, without the need for rigid reaction conditions or unjustifiably large amounts of the catalyst.

12 cl, 2 ex

FIELD: chemistry.

SUBSTANCE: invention relates to a novel method of producing halogen-substituted aromatic amines. The method includes hydrogenating halogen-substituted aromatic nitro-compounds in an isopropanol medium in the presence of aluminium oxide as a heterogeneous catalyst. The process is carried out by adding supercritical isopropyl alcohol at temperature of 250-340°C and pressure of 150-220 atm.

EFFECT: conducting the process in said conditions considerably shortens reaction time (less than 6 minutes instead of 3,5-6 hours), does not require use of expensive catalysts and enables to obtain products with high selectivity, output and conversion.

2 cl, 5 tbl, 4 ex

FIELD: chemistry.

SUBSTANCE: specified flows of gaseous hydrogen and liquid aromatic nitrocompound are supplied into specified input side. Specified gaseous hydrogen and specified aromatic nitrocompound are converted into aromatic amine, by which output products of reactor, containing said aromatic amine and water, are provided. Said output products of reactor are discharged from reactor on output side of said reactor. Method is characterised by the fact that water is supplied into input side of reactor with molar ratio of the number of water moles to the number of hydrogen moles being in the interval from 1.5 to 7.5. Claimed method makes it possible to reduce amount of byproducts in output products of reactor.

EFFECT: providing flow of gaseous hydrogen and flow of liquid aromatic nitrocompound, providing catalytic reactor with immobile layer, which has input and output sides.

5 cl, 3 dwg, 2 tbl

Chemical plant // 2508287

FIELD: chemistry.

SUBSTANCE: chemical plant includes a first process unit, having a reactor for producing nitrobenzene via nitration of benzene to produce a first waste water stream containing nitrobenzene; at least a second process unit, having a reactor for producing aniline by reducing nitrobenzene with hydrogen to provide a second waste water stream containing aniline; an aniline purification apparatus for removing nitrobenzene from aniline, a stripping column for separating aniline from the waste water stream. The first waste water stream is fed into the stripping column below the point of input of the second waste water stream, where said stripping column separates aniline and nitrobenzene from said first and second waste water streams, and the separated aniline and nitrobenzene are fed into said aniline purification apparatus. The stripping column normally used for the plant has n theoretical trays denoted by symbols A1 through An, wherein A1 corresponds to the top tray and An corresponds to the bottom tray; said second waste water stream is fed into the stripping column at theoretical tray Ax, where x is not less than 1, and said first stream is fed into the stripping column at theoretical tray A[y], where [y] is the integer part of the value y, and the value y itself satisfies the equation 0.5*(n+1)+0.5*x<y<0.85*(n+1)+0.15*x. The plant can further include a reactor for producing diaminodiphenylmethane (DADPM) for converting said aniline of said aniline stream into DADPM.

EFFECT: invention enables to obtain products of high quality with a considerable economic effect by avoiding the need to treat each stream separately.

18 cl, 2 dwg, 2 tbl

FIELD: chemistry.

SUBSTANCE: invention relates to an improved method of producing N-alkyl-N'-n-phenylenediamines of general formula 1, where: R1, R2 are alkyl substitutes. The method involves reducing alkylation of 4-nitrodiphenylamine (4-NDPA) with aliphatic ketones of general formula R1-CO-R2, where R1, R2 are C1-C10 alkyl substitutes, in the presence of hydrogen deposited on a granular carbon support of a palladium catalyst Pd/C and a monobasic aliphatic carboxylic acid R"COOH, where: R"=CnH2n+1, n=1-4, in the medium of a solvent - monoatomic saturated alcohol R'OH, where; R' is a C3-C20 alkyl substitute. Content of palladium in the supported catalyst is equal to 0.4-0.6 wt %. The process is usually carried out at temperature of 50-190°C. The molar ratio of catalyst (with respect to Pd) to 4-NDPA ranges from 0.05:1 to 0.1:1, molar ratio of 4-NDPA to the aliphatic ketone is 1:5-1:10, molar ratio of the monobasic aliphatic carboxylic acid to 4-NDPA is 0.036:1-0.1:1 and molar ratio of 4-NDPA to the solvent is 0.25:1 -0.4:1. Hydrogen pressure in the process is equal to 40-60 atm. The obtained N-alkyl-N'-phenyl-n-phenylenediamines have general formula 1

,

where: R1, R2 are alkyl substitutes. The method simplifies the process by reducing the amount of precious metals and improves separation of products at the end of the process. High output of the end product and selectivity of the process are preserved.

EFFECT: high quality with high gas chromatography purity.

7 cl, 3 ex

FIELD: chemistry.

SUBSTANCE: invention relates to a novel method of producing 2-(aminoalkyl)-3-(aminophenyl)bicyclo[2.2.1]heptanes of formula (I). Mono- and diamines containing a bicyclic moiety can be used in medicine as an agent for medicinal preparations having thymoleptic, tonic action, are used in treating cocaine addiction and can also be of interest as potential monomers for polycondensation polymers. The method of producing said compounds involves reacting a nitrocinnamic aldehyde (dienophile) with cyclopentadiene to obtain a corresponding 3-(nitrophenyl)bicyclo[2.2.1]hept-5-ene-2-carbaldehyde, which is condensed with proton-mobile compounds, for example with hydroxyl amine, nitromethane or cyanoacetic acid. The corresponding 2-substituted-3-phenylbicyclo[2.2.1]hept-5-ene derivative is obtained, which is reduced with active hydrogen obtained by reacting a nickel-aluminium alloy with potassium hydroxide to obtain a compound of general formula (I) where n=1, 2, 3.

EFFECT: use of the disclosed method as a source compound of nitrocinnamic aldehyde enables to obtain a product with high degree of purity and output, simplifies the process and improves technological effectiveness thereof, reduces reactivity of compounds in Diels-Alder reactions, enables to obtain not only aminomethyl compounds, but also other aminoalkyl compounds using a simpler process.

1 dwg, 8 ex

Up!