Antiviral compounds based on condensed dihydroxyisochromene-naphthoimidazoles

Hepatitis C is recognized as a chronic viral disease of the liver, which is characterized by liver disease. Although liver-targeting drugs are widely used and have shown their efficacy, toxicity and other side effects have limited their usefulness. Hepatitis C virus (HCV) inhibitors are useful in limiting the establishment and evolution of HCV infection, as well as in HCV diagnostic tests.

There is a need for new HCV therapeutic agents. In particular, there is a need for HCV therapeutic agents that have broad activity against HCV genotypes (eg genotypes at, lb, 2a, 3a, 4a). There is also a need for drugs that are less sensitive to viral resistance. Inhibitor resistance mutations have been described for HCV NS5A for genotypes at and lb in [1]. '

The present invention describes a compound of the formula (I) generated:

О or о a pharmaceutically acceptable salt or a prodrug thereof.

The present invention also provides a pharmaceutical composition comprising a pharmaceutically acceptable compound or salt as described below and at least one pharmaceutically acceptable carrier.

In one embodiment, as described below, the composition further contains a nucleoside or nucleotide inhibitor of HCV NS5B polymerase.

The present invention provides a pharmaceutically acceptable compound or salt, as described below, for use in the treatment of hepatitis C.

In one embodiment, the pharmaceutically acceptable composition or salt is provided for use as described below in combination with a nucleoside or nucleotide inhibitor of HCV NS5B polymerase.

In one embodiment, the composition comprises an additional therapeutic agent for treating HCV. In one embodiment the therapeutic agent is selected from ribavirin, an NS3 protease inhibitor, a nucleoside or nucleotide inhibitor of HCV NS5B polymerase, an alpha-glucosidase 1 inhibitor, a hepatoprotectant, non-nucleoside inhibitors of VHC NS5B polymerase or combinations of them. In one embodiment, the additional compound further contains the nucleoside or nucleotide inhibitor of HCV NS5B polymerase. In one embodiment, the nucleoside or nucleotide inhibitor of HCV NS5B polymerase is selected from ribavirin, viramidine, levovirine, O-nucleoside or isatoribine.

In one embodiment, the pharmaceutical composition comprising a compound as described herein and at least one nucleoside or nucleotide inhibitor of HCV NS5B polymerase, and at least one pharmaceutically acceptable carrier, is provided. In one embodiment, the composition further comprises an interferon, a pegylated interferon, о ribavirin or combinations thereof. In one embodiment, the compound is a compound exemplified in Example PY. In one embodiment, the nucleoside or nucleotide inhibitor of HCV NS5B polymerase is sofosbuvir.

In one embodiment the present invention also provides a pharmaceutical composition which further contains a pegylated interferon or interferon.

In one embodiment the present invention also provides a pharmaceutical composition which further contains a nucleoside analog.

In one embodiment, the present invention also provides a pharmaceutical composition wherein the nucleoside analogue is selected from ribavirin, viramidine,

MD 4403 Cl 2016.09.30 levovirine, о L-nucleoside and isatoribine, and interferon is cc-interferon or pegylated ccinterferon.

In one embodiment, the present invention also provides a compound of the invention for use in medical therapy (for example, for use in inhibiting HCV activity or treating a disease associated with HCV activity).

The present invention also provides novel synthetic processes and intermediates according to the invention which are useful for the preparation of the compounds of the invention. Some of the compounds of the invention are useful for the preparation of other compounds of the invention.

In another aspect, the invention relates to a compound of the invention, or to a pharmaceutically acceptable salt thereof, for use in the prophylactic or therapeutic treatment of hepatitis C or an associated hepatitis C disorder.

Compounds of formula (I) have been shown to possess useful activity against HCV genotypes 1. In addition, certain compounds of formula (I) have significant potency over resistant strains in GT1.

Accordingly, certain compounds of formula (I) possess beneficial pharmacological properties that make them useful for meeting the current need for HCV agents with such beneficial properties.

In one embodiment, the invention provides a compound having improved inhibitory or pharmacokinetic properties, including increased activity against viral resistance development, improved oral bioavailability, higher potency (e.g., inhibition of HCV activity), or extended half-life in vivo. Certain compounds of the invention may have fewer side effects, less complicated dosage regimens, or may be orally active.

The reference will now be detailed in detail to certain embodiments of the invention, examples of which are illustrated in the accompanying structures and formulas. As the invention will be described in connection with the embodiments listed, it will be understood that they do not intend to limit the description to such embodiments only. On the contrary, the invention is intended to cover all alternatives, modifications and equivalents, which may be included within the scope of the present invention as defined by the embodiments.

"Alkyl" means C1-C18 hydrocarbon containing normal, tertiary or cyclic, secondary, carbon atoms. Examples are methyl (Me, -CH₃), ethyl (Et, -CH₂CH₃), 1-propyl (n-Pr, n-propyl, -CH2CH2CH3), 2-propyl (i-Pr, i-propyl, -CH (CH)₃)₂), 1-butyl (n-Bu, n-butyl, -CH₂CH₂CH₂CH₃), 2-methyl-1-propyl (i-Bu, i-butyl, -CH₂(CH₃)₂), 2-butyl (s-Bu, s-butyl, -CH (CH₃) CH₂CH₃), 2-methyl-2-propyl (t-Bu, t-butyl, -C (CH₃)₃), 1-pentyl (n-pentyl, -CH₂CH₂CH₂CH₂CH₃), 2-pentyl (-CH (CH₃) CH₂CH₂CH₃), 3-pentyl (-CH (CH₂CH₃)₂), 2-methyl-2-butyl (-C (CH₃)₂CH₂CH₃), 3-methyl-2-butyl (-CH (CH₃) CH (CH₃)₂), 3-methyl-1-butyl (CH₂CH₂CH (CH₃)₂), 2-methyl-1-butyl (-CH₂CH (CH₃) CH₂CH₃), 1 -hexyl (CH₂CH₂CH₂CH₂CH₂CH₃), 2-hexyl (-CH (CH₃) CH₂CH₂CH₂CH₃), 3-hexyl (CH (CH₂CH₃) (CH₂CH₂CH₃)), 2-methyl-2-pentyl (-C (CH₃)₂CH₂CH₂CH₃), 3-methyl-2pentyl (-CH (CH₃) CH (CH₃) CH₂CH₃), 4-methyl-2-pentyl (-CH (CH₃) CH₂(CH₃)₂), 3-methyl-3pentyl (-C (CH₃) (CH₂CH₃)₂), 2-methyl-3-pentyl (-CH (CH₂CH₃) CH (CH₃)₂), 2,3-dimethyl-2-butyl (-C (CH₃)₂CH (CH₃)₂), 3,3-dimethyl-2-butyl (-CH (CH₃) C (CH₃)₃, and cyclopropylmethyl.

"Alkenyl" means о hydrocarbon C₂-But₈ containing secondary, normal, tertiary or cyclic carbon atoms with at least one unsaturation center, that is, carbonyl carbon bond, sp² double. Examples include, but are not limited to, ethylene or vinyl (CH = CH₂), others (-CH₂CH = CH₂), cyclopentenyl (-C₅H₇), and 5-hexeml (-CH₂ CH₂CH₂CH₂CH = CH₂).

"Alkynyl" means reprezintă C hydrocarbon₂-But₈ which contains secondary, normal, tertiary or cyclic carbon atoms with at least one unsaturation center, i.e., carbonyl carbon bond, triple sp. Examples include, but are not limited to, acetylene (-C = CH) and propargyl (-CH) groups.₂C = CH).

MD 4403 Cl 2016.09.30 "Alkylene" refers to a saturated, linear or branched chain or a cyclic hydrocarbon radical with 1-18 carbon atoms, and having two monovalent radical centers obtained by removing two hydrogen atoms from the same or two different carbon atoms of a parent alkane. Typical alkylene radicals include, but are not limited to, methylene (-CH₂-) 1,2-ethyl (-CH₂CH₂), 1,3-propyl (-CH₂CH₂CH₂), 1,4-butyl (-CH₂CH₂CH₂CH₂) etc.

"Alkenylene" refers to an unsaturated, branched or straight chain or a cyclic hydrocarbon radical with 2-18 carbon atoms, and having two monovalent radical centers obtained by removing two hydrogen atoms from the same or two different carbon atoms. of a parental alkene. Typical alkenylene radicals include, but are not limited to, 1,2-ethylene (-CH = CH-).

"Alkylene" refers to an unsaturated, branched or straight chain or a cyclic hydrocarbon radical with 2-18 carbon atoms, and having two monovalent radical centers obtained by removing two hydrogen atoms from the same or two different carbon atoms of of a parental alkina. Typical alkynylene radicals include, but are not limited to, acetylene (-OC-), propargyl (-CH)₂OC-), and 4-pentinyl (-CH₂CH₂CH₂C = CH).

The term "alkoxy" or "alkyloxy", as used herein, refers to the alkyl group attached to the initial molecular moiety through an oxygen atom.

The term "alkoxycarbonyl", as used herein, refers to the alkoxy group attached to the unipolar molecular moiety through a carbonyl group.

The term "cycloalkyl", as used herein, refers to a monocyclic saturated hydrocarbon ring system having from three to seven carbon atoms and zero heteroatoms. Representative examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclopentyl and cyclohexyl. The cycloalkyl groups of the present invention are optionally substituted with one, two, three, four or five substituents independently selected from alkoxy, alkyl, aryl, cyano, halo, haloalkoxy, haloalkyl, heterocyclyl, hydroxy, hydroxyalkyl, nitro and -NR^xR^y, wherein the aryl and heterocyclyl are further optionally substituted with one, two or three substituents independently selected from alkoxy, alkyl, cyano, halo, haloalkoxy, haloalkyl, hydroxy and nitro.

The term "cycloalkylcarbonyl", as used herein, refers to the cycloalkyl group attached to the initial molecular moiety through a carbonyl group.

The term "cycloalkyloxy", as used herein, refers to the cycloalkyl group attached to the initial molecular moiety through an oxygen atom.

The term "cycloalkyloxycarbonyl", as used herein, refers to the cycloalkyloxy group attached to the initial molecular moiety through a carbonyl group.

By "aryl" is meant a monovalent aromatic hydrocarbon radical with 6-20 carbon atoms obtained by removing a hydrogen atom from a single carbon atom of a paternal aromatic ring system. Typical aryl groups include, but are not limited to, benzene derivatives, substituted benzene, naphthalene, anthracene, biphenyl, and the like.

"Arylalkyl" refers to an acyclic alkyl radical, m which one of the hydrogen atoms binds to a carbon atom, usually a terminal carbon atom or sp³, is replaced by an aryl radical. Typical arylalkyl groups include, but are not limited to, benzyl, 2-phenylethan-1-yl, naphthylmethyl, 2-naphthylethan-1-yl, naphthobenzyl, 2-naphthophenylethan-1-yl and the like. The arylalkyl group comprises from 6 to 20 carbon atoms, for example, the alkyl moiety, including the alkanyl, alkenyl or alkynyl groups, of the arylalkyl group having from 1 to 6 carbon atoms and the aryl moiety having from 5 to 14 carbon atoms. .

"Substituted alkyl", "substituted aryl" and "substituted arylalkyl" means alkyl, aryl and arylalkyl, respectively, wherein one or more hydrogen atoms are each independently replaced by a non-hydrogen substituent. Typical substituents include, but are not limited to: halo (eg, F, Cl, Br, I), -R, -OR, -SR, -NR₂, -CF₃, -CC1₃, -OCF₃, -CN, -NO₂, -N (R) C (= O) R,

-C (= O) R, -OC (= O) R, -C (O) OR, -C (= O) NRR, -S (= O) R, -S (= O)₂OR, -S (= O)₂R, OS (= O)₂OR

-S (= O)₂NRR, and each R is independently -H, alkyl, aryl, arylalkyl or heterocycle. The alkylene, alkenylene and alkynylene groups may also be similarly substituted.

MD 4403 Cl 2016.09.30

The term "optionally substituted" with reference to a particular fragment of the compound of formula I, (for example, optionally substituted aryl group) refers to a fragment having 0, 1, 2 or more substituents.

The symbol "-----" in an annular structure means that о bond is о single or double bond. in a non-limiting example,

<img file="MD4403C1_D0001.tif" />

The term "haloalkyl", as used herein, includes о alkyl group substituted with one or more halogens (eg, F, Cl, Br or I). Representative examples of haloalkyl include trifluoromethyl, 2,2,2-trifluoroethyl and 2,2,2-trifluoro-1- (trifluoromethyl) ethyl.

The "heterocycle" or "heterocyclyl" as used herein includes, for example, but without limitation these heterocycles described in [2], [3], [4]. In a specific embodiment, the "heterocycle" includes a "carbocycle", as defined herein, where one or more (e.g., 1, 2, 3 or 4) carbon atoms have been replaced by a heteroatom. (for example, Ο, N or S). The term, "Jieterocycle" also includes "heteroaryl", which is a heterocycle, wherein at least one of the heterocyclic nuclei is aromatic.

Examples of heterocycles include, but are not limited to, pyridyl, dihydropyridyl, tetrahydropyridyl (piperidyl), thiazolyl, tetrahydrothiophenyl, tetrahydrothiophenyl sulfate oxidized, pyrimidinyl, furanyl, thienyl, pyrolyl, pyrazolyl, benzidurol, imidazolyl, imidazolyl quinolinyl, isoquinolinyl, benzimidazolyl, piperidinyl, 4-piperidonyl, pyrrolidinyl,

2-pyrrolidonyl, pyrolinyl, tetrahydrofuranyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, octahydroisoquinolinyl, azocinyl, triazinyl, 6H-1, 2,5-thiadiazinyl, 2H, 6H-1, 5,2-dithiazinyl, thienyl, pyridyl, thienyl, pyridyl, thienyl, pyridyl , chromenyl, xanthenyl, phenoxatinyl, 2H-pyrrolyl, isothiazolyl, isoxazolyl, pyrazinyl, indolizinyl, isoindolyl, 3Hindolyl, 1H-indazolyl, purinyl, 4H-quinolizinyl, phthalazinyl, naphthyridinyl, chinoxynolin, quinolinyl, quinolinyl, quinoline 4H-carbazolyl, carbazolyl, β-carbolinyl, phenanthridinyl, acridinyl, pyrimidinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, furazanyl, phenoxazinyl, isochromanyl, cromanyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, pyrazolinyl, pyrazoline benzotriazolyl, benzisoxazolyl, oxindolyl, benzoxazolinyl, isatinoyl and bis-tetrahydrofuranil:

<img file="MD4403C1_D0002.tif" />

For example, but without limitation, carbon-linked heterocycles are bound at position 2, 3, 4, 5 or 6 of a pyridine, position 3, 4, 5 or 6 of a pyridazine, position 2, 4, 5 or 6 of a pyridine. pyrimidines, position 2, 3, 5 or 6 of a pyrazine, position 2, 3, 4 or 5 of a furan, tetrahydrofuran, thiofuran, thiophene, pyrrole or tetrahydropyrol, position 2, 4 or 5 of an oxazole, imidazole or thiazole, position 3, 4 or 5 of an isoxazole, pyrazole or isothiazole, position 2 or 3 of an aziridine, position 2, 3 or 4 of an azetidine, position 2, 3, 4, 5, 6, 7 or 8 of a quinoline or position 1, 3, 4, 5, 6, 7 or 8 of an isoquinoline. More specifically, carbon-linked heterocycles include 2-pyridyl, 3-pyridyl, 4-pyridyl, 5-pyridyl, 6-pyridyl, 3-pyridazinyl, 4-pyridazinyl, 5-pyridazinyl, 6-pyridazinyl, 2-pyrimidinyl, 4-pyrimidinyl, 5 -pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl, 3-pyrazinyl, 5-pyrazinyl, 6-pyrazinyl, 2-thiazolyl, 4-thiazolyl or 5-thiazolyl.

For example, but without limitation, nitrogen-linked heterocycles are linked to each other at position 1 of aziridine, azetidine, pyrrole, pyrrolidine, 2-pyrrole, 3-pyrrole, imidazole, imidazolidine, 2-. imidazoline, 3-imidazoline, a pyrazole, a pyrazoline, 2-pyrazoline, 3-pyrazoline, a piperidine, a piperazine, an indole, a linen, a

MD 4403 Cl 2016.09.30

IH-indazole, position 2 of an isoindole or isoindoline, position 4 of a morpholine and position 9 of a carbazole or β-Carboline. More specifically, nitrogen-linked heterocycles include

1-aziridyl, 1-azetethyl, 1-pyrrolyl, 1-imidazolyl, 1-pyrazolyl and 1-piperidinyl.

"Carbocycle" refers to a saturated, unsaturated or aromatic ring having up to about 25 carbon atoms. Typically, a carbocycle has about 3 to 7 carbon atoms as a unicycle, about 7 to 12 carbon atoms as a bicycle, and up to about 25 carbon atoms as a polycycle. Monocyclic carbocycles usually have from 3 to 6 ring atoms, and more specifically 5 or 6 ring atoms. Bicyclic carbocycles typically have from 7 to 12 ring atoms, for example, arranged as a bicyclic system [4,5], [5,5], [5,6] or [6,6], or 9 or 10 ring atoms arranged as a bicyclic system [5,6] or [6,6]. The term carbocycle includes "cycloalkyl", which is a saturated or unsaturated carbocycle. Examples of monocyclic carbocycles include cyclopropyl, cyclobutyl, cyclopentyl, I-cyclopent-I-enyl, l-cyclopent-2-enyl, l-cyclopent-3-enyl, cyclohexyl, I-cyclohex-I-enyl, l-cyclohex-2-enyl , cyclohex-3-enyl, phenyl, spiryl and naphthyl.

The term "amino", as used herein, refers to -NH₂.

The term "chiral" refers to molecules that have the non-superimposibility property of the mirror-structure, while the term "achiral" refers to molecules that are superimposed on their mirror-structure.

The term "stereoisomers" refers to compounds that have identical chemistry, but differ in the arrangement of atoms or groups in space.

"Diastereomer" refers to a stereoisomer with two or more centers of chirality and whose molecules are not mirror images of another. Diastereomers have different physical properties, for example, melting points, boiling points, spectral properties and reactivities. Mixtures of diastereomers can be separated by high-resolution analytical procedures, such as electrophoresis and chromatography.

"Enantiomers" refers to two stereoisomers of one compound, which are non-superimposed mirror images of another.

The term "treatment" or "treatment", insofar as it refers to a disease or condition, includes the prevention of the occurrence of the disease or condition, the inhibition of the disease or condition, the elimination of the disease or condition, and / or the alleviation of one or more symptoms of the disease or condition. .

The stereochemical definitions and conventions used herein are generally compliant with sources [5] and [6]. There are many organic compounds in optically active forms, that is, they have the ability to rotate the polarized light density. When describing an optically active compound, the prefixes (D and L) or (R and S) are used to indicate the absolute configuration of the molecule around its chiral center (s). The prefixes d and 1 or (+) and (-) are used to designate the rotating sign of the plane polarized light by the compound, with (-) or 1 meaning that the compound is levogir. A compound prefixed with (+) or d is dextrogir. For some chemical structure, these stereoisomers are identical except that they are mirror images of another. A specific stereoisomer may also be referred to as an enantiomer, and a mixture of such isomers is often referred to as a mixture of enantiomers. A 50:50 mixture of enantiomers is referred to as a racemic mixture or a racemate, which may occur if there was no stereo hepatic or stereospecificity in a chemical reaction or chemical process. The terms "racemic mixture" and "racemate" refer to an equimolar mixture of two enantiomeric species, lacking optical activity. The invention includes all stereoisomers of the compounds described herein.

prodrugs

The term "prodrug", as used herein, refers to any compound that when administered to a biological system generates a compound of the invention that inhibits HCV activity (the "active inhibitory compound"). The compound may be comprised of the prodrug as a result of: (i) spontaneous chemical reaction (s), (ii) enzyme-catalyzed chemical reaction (s), (iii) photolysis and / or (iv) metabolic chemical reaction (s).

The "prodrug fragment" refers to the labile functional group which is separated from the active inhibitory compound during metabolism, systemically, within a cell, by hydrolysis, enzymatic cleavage or by another process [7] [8]. Enzymes that are capable of an enzymatic activation mechanism with prodrug compounds

MD 4403 Cl 2016.09.30 described in the invention include, but are not limited to, amidases, esterases, microbial enzymes, phospholipases, cholinesterases and phosphates. The prodrug fragments can contribute to increased solubility, absorption and lipophilicity to optimize drug delivery, bioavailability and efficacy. The fraction of the prodrug may include an active metabolite or the drug itself.

Exemplary fragments of the prodrug include hydrolytically sensitive or labile acyloxymethyl esters - CH₂OC (= O) R⁹⁹ and acyloxymethylcarbonate - CH2OC (= O) OR⁹⁹ where R⁹⁹ is a C 1 -C 6 alkyl group, C 1 -C 8 substituted alkyl, C 1 -C 2 2 aryl or C 1 -C substituted aryl₂₀. The acyloxyalkyl ester was first used as a strategic о of the prodrug for carboxylic acids and then applied to phosphates and phosphonates by Farquhar and others [9], as well as patents [10], [И], [12], [13 ]. Subsequently, acyloxyalkyl ester was used to fumigate phosphonic acids through cell membranes and enhance oral bioavailability. A variant close to the acyloxyalkyl ester, the alkoxycarbonyloxyalkyl ester (carbonate), may also promote oral bioavailability as a fragment of the prodrug in the compounds of the combinations described in the present invention. An exemplary acyloxymethyl ester is pivaloyloxymethoxy, (POM) CH₂OC (= O) C (CH₃)₃. An exemplary fragment of the acyloxymethylcarbonate prodrug is pivaloyloxymethylcarbonate (РОС) - CH₂OC (= O) OC (CH₃)₃.

Aryl esters of phosphorus groups, especially phenyl esters, are reported to increase oral bioavailability [14]. Phenyl esters containing a carboxylic ester in the ortho-phosphate position have also been described [15]. Benzyl esters are reported as generating initial phosphonic acids. In some cases, the substitutes in the ortho- or parapot position accelerate hydrolysis. Benzyl analogues with an acylated phenol or an alkylated phenol can generate the phenolic compound by the action of enzymes, for example esterases, oxidases, etc., which in turn are subjected to cleavage of the C-0 benzyl bond to generate phosphoric acid and a methyl intermediate quinones. Examples of this class of prodrugs are described by Mitchell et al. [16], [17]. Other benzyl prodrugs have been described, which contain a group containing carboxylic ester attached to benzyl methylene [17]. Confined prodrugs are reported to be useful for intracellular delivery of phosphonated drugs. These progenitors confine the ethylthio group in which the thiol group is either esterified by an acyl group or combined with another thiol group to form disulfide. Deesterification or reduction of disulfide generates the free thiointermediate which subsequently decomposes to phosphoric acid and episulfide [18], [19].

Protective groups

In the context of the present invention, protecting groups include fragments of the prodrug and chemical protecting groups.

"Protective group" refers to a fragment of a compound that hides or modifies the properties of a functional group or the properties of the compound as a whole. Chemical protection groups and protection / deprotection strategies are well known in the art. See, for example, [20]. Protective groups are often used to mask the reactivity of certain functional groups, to enhance the efficiency of the desired chemical reactions, for example, making and breaking chemical bonds in a given and planned manner. The protection of functional groups of a compound modifies other physical properties besides the reactivity of the protected functional group, such as polarity, lipophilicity (hydrophobicity) and other properties that can be measured by common analytical tools. Chemically protected intermediates may be themselves biologically active or inactive.

Protected compounds may also exhibit modified and, in some cases, optimized properties in vitro and in vivo, such as through cell membranes and resistance to enzymatic degradation or sequestration. In this role, the protective compounds with intended therapeutic effects can be menfionafi as prodrugs. Another function of a protective group is to convert the parent drug into a prodrug, whereby the parent drug is released upon conversion of the prodrug in vivo. Because the active prodrugs can be absorbed more effectively than the parent drug, the prodrugs may have a higher potency in vivo than the parent drug. Protective groups are evacuated either in vitro, in the case of chemical intermediates, or in vivo, in the case of prodrugs. With regard to chemical intermediates, it is not particularly important that products resulting from deprotection, for example, alcohol,

MD 4403 Cl 2016.09.30 to be physiologically acceptable, although, in general, it is more desirable if the products are pharmacologically harmless.

Protective groups are available, known and commonly used, and are optionally used to prevent side reactions with the protected group during synthetic procedures, ie the processes or methods of preparing the compounds of the invention. For most, the decision according to which grouping to protect, when necessary, the nature of the "GP" chemical protecting group will be dependent on the reaction chemistry to be protected against (for example, acidic, basic, oxidative, reductive or other conditions) and the desired direction of synthesis. GPs should not be, and generally are not, identical if the compound is substituted with multiple GPs. Generally, GPs will be used to protect functional groups such as carboxyl, hydroxyl, thio or amino groups and thus prevent adverse reactions or otherwise facilitate synthetic efficacy. The order of deprotection to obtain the free unprotected groups depends on the desired direction of the synthesis and the conditions of the reaction to be collided, and may occur in any order as determined by the craftsman.

Different functional groups of the compounds of the invention can be protected. For example, protecting groups for -OH groups (either hydroxyl, carboxylic acid, phosphonic acid or other functions) include "groups which form ethers- or esters". The ester-ester groups are capable of functioning as chemical protecting groups in the synthetic schemes provided herein. However, some hydroxyl and thio protecting groups are neither ether-forming groups nor ester-forming groups, as will be understood by those skilled in the art, and are included with the amides described below.

A very large number of hydroxyl protecting groups and amide-forming groups and the corresponding chemical cleavage reactions are described in [21]. See also [22], included herein by reference in its entirety. In particular [23], [24], [25], [26], [27]. For carboxylic acid, phosphonic acid, sulfonic acid, phosphonate and other acid protecting groups see Greene, as described below.

For example, but without limitation, the variables described herein may be recursive substituents in certain embodiments. Typically, each of these can occur independently of 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 times in a certain embodiment. Specifically, each of these can occur independently of 12 or more times or in a certain variant. Whenever a compound described in this document is substituted by more than one of the same designated group, for example, "R¹"Or" R³"Then it will be understood that the groups can be the same or different, for example, each group is chosen independently. The wavy lines indicate the site of covalent bonds to adjacent, fragmented or atoms groups.

In one embodiment of the invention, the compound is in an isolated and purified form. In general, the term "isolated and purified" means that the compound is substantially free of biological materials (eg, blood, tissues, cells, etc.). In a specific embodiment of the invention, the term means that the compound or conjugate according to the invention is at least about 50% by weight without biological materials; In another specific embodiment, the term means that the compound or conjugate of the invention is at least about 75% by weight without biological materials; In another specific embodiment, the term means that the compound or conjugate according to the invention is at least about 90% by weight without biological materials; In another specific embodiment, the term means that the compound or conjugate of the invention is at least about 98% by weight without biological materials. And in another embodiment, the term means that the compound or conjugate according to the invention is at least about 99% by weight without biological materials. In another specific embodiment, the invention provides a compound or conjugate that has been prepared. synthetically (eg ex vivo).

stereoisomers

The compounds of the invention may have chiral centers, for example, chiral carbon or phosphorus atoms. The compounds of the invention thus include racemic mixtures of all stereoisomers, including enantiomers, diastereoisomers and atropisomers. In addition, the compounds according to the invention include enriched optical isomers or soluble at

MD 4403 Cl 2016.09.30 any or all of the asymmetric, chiral atoms. In other words, the apparent chiral centers in the descriptions are presented as chiral isomers or racemic mixtures. Both racemic mixtures and diastereomeric mixtures as well as isolated or synthesized individual optical isomers, substantially free of enantiomeric structures or diastereomer ice, are all within the scope of the invention. Racemic mixtures are separated into their substantially optically pure individual isomers by well-known methods, such as, for example, separation of diastereomeric salts formed with optically active adjuvants, for example acids or bases, followed by conversion back to optically active substances. In most cases, the desired optical isomer is synthesized by stereospecific reactions, starting with the corresponding stereoisomer of the desired starting material.

The compounds, according to the invention, may also exist as tautomeric isomers in certain cases. Although only one tautomer can be described, all these forms are contemplated within the scope of the invention. For example, enamine amine tautomers may exist for purine, pyrimidine, imidazole, guanidine, amidine, and tetrazole systems and all their possible tautomeric forms are included in the scope of the invention.

Salts and hydrates

Examples of physiologically or pharmaceutically acceptable salts of the compounds of the invention include salts derived from a suitable base, such as an alkali metal (e.g., sodium), an alkaline earth metal (e.g., magnesium), ammonium, and NX / (where X represents о C 1 -C 6 alkyl group₄). Physiologically acceptable salts of a hydrogen atom or an amino group include salts of organic carboxylic acids, such as acetic, benzoic, lactic, fumaric, tartaric, maleic, malonic, malic, isetionic, lactobionic and succinic acids; organic sulfonic acids such as methanesulfonic, ethanesulfonic, benzenesulfonic and p-toluenesulfonic acid; and inorganic acids such as hydrochloric, sulfuric, phosphoric and sulfamic acids. Physiologically acceptable salts of a hydroxy group compound include the anion of the mentioned compound in combination with a suitable cation, such as Na⁺ and NX / (wherein X is independently selected from H or о C1-C6 alkyl group₄).

For therapeutic use, the salts of active ingredients of the compounds according to the invention will usually be physiologically acceptable, ie they will be salts derived from a physiologically acceptable acid or base. However, salts of acids or bases which are not physiologically acceptable may also be used, for example, in the preparation or purification of a physiologically acceptable compound. All salts, whether or not derived from a physiologically acceptable acid or base, are included in the scope of the present invention.

The metal salts are usually prepared by reacting the metal hydroxide with a compound of this invention. Examples of metal salts, which are thus prepared, are the salts containing L /, Na⁺ and the more soluble puffer metal salt may be precipitated from the solubility of a more soluble salt by addition of the appropriate metal compound.

In addition, salts may be formed from the acid addition of certain organic and inorganic acids, for example, HCl, HBr, H₂SO₄, H₃PO₄ or organic sulfonic acids, at the base centers, usually the amines, or the acid groups. Finally, it is understood that the compositions described herein include compounds of the present invention in their non-ionized form, such as amphoteric, and combinations with stoichiometric quantities of water as well as in hydrates.

Also included in the scope of this invention are salts of parent compounds with one or more amino acids. Any of the natural or unnatural amino acids are suitable, especially amino acids that occur naturally as proteins, although the amino acid is usually one that carries a side chain with a basic or acidic group, for example lysine, arginine or glutamic acid, or о neutral group such as glycine, serine, threonine, alanine, isoleucine or leucine.

HCV inhibition methods

Another aspect of the invention relates to methods for inhibiting HCV activity comprising the step of treating a sample suspected of confining HCV with a compound or о composition of the present invention.

The compounds according to the invention may act as HCV inhibitors, as intermediates for such inhibitors or have other utilities as described below. Inhibitors will generally bind to pores on the surface or in a liver cavity. The compounds that bind in the liver can bind with different degrees of reversibility. Those compounds that bind

MD 4403 Cl 2016.09.30 substantially irreversible are the ideal candidates for use in this method of the present invention. Once labeled, compounds that bind substantially irreversibly are useful as probes for HCV detection. Accordingly, the invention relates to methods of detecting NS3 in a suspected HCV boundary sample comprising the steps of: treating a suspected sample containing HCV with a composition comprising a compound of the invention bound to the label; and observing the effect of the sample on the activity of the label. Suitable labels are well known in the field of diagnostics and include radicals! free stable, fluorophores, radioisotopes, enzymes, chemiluminescent and chromogenic groups. The compounds described herein are conventionally labeled using functional groups, such as amino or hydroxyl. In one embodiment, the invention describes a compound of any of the formulas (I) and (Al) - (A4), which comprises or is linked or coupled to one or more detectable labels. In the context of the invention, samples suspected of containing HCV include natural or artificial materials, such as living organisms; tissue or cell cultures; biological samples, such as biological material samples (blood, serum, urine, cerebrospinal fluid, tears, sputum, saliva, tissue samples and the like); laboratory tests; food, water or air samples; bioproduct samples such as cell extracts, recombinant cells specifically synthesizing the desired glycoprotein; and the like. Usually the sample will be suspected of confining HCV. Samples can be confined to any medium, including water and organic solvent / water mixtures. Samples include living organisms such as humans, as well as artificial materials such as cell cultures.

The treatment step according to the invention includes adding the compound of the invention to the sample or this comprises adding a precursor of the composition to the sample. The additional step includes any method of administration as described above.

If desired, HCV activity after compound application can be observed by any method, including direct and indirect methods of detecting HCV activity. Quantitative, qualitative and semi-quantitative methods for determining HCV activity are all considered. Usually one of the screening methods described above is applied, however, any other method, such as observing the physiological properties of an HIV organism are also applicable.

Many organisms border HCV. The compounds of the present invention are useful in the treatment or prophylaxis of disorders associated with HCV activation in animals or humans.

However, in screening compounds capable of inhibiting HCV activity, it should be taken into account that the results of enzyme tests may not always be correlated with cell culture assays. Thus, a cell-based assay would typically be primary screening instrumentation.

Pharmaceutical compositions (formulations)

The compounds of the present invention are formulations with conventional carriers and excipients, which will be selected in accordance with ordinary practice. The tablets will contain excipients, gliders, fillers, binders and the like. The aqueous compositions are prepared in sterile form, and when intended for delivery by administration other than oral administration will generally be isotonic. All compositions will optionally contain excipients, such as those provided in [27]. Excipients include ascorbic acid and alpha antioxidants, chelating agents such as EDTA, carbohydrates, such as dextrin, hydroxyalkyl cellulose, hydroxyalkylmethyl cellulose, stearic acid and the like. The pH of the compositions ranges from about 3 to about 11, but is usually about 7 to 10. Typically, the compound will be administered at a dose of 0.01 milligrams to 2 grams. In one embodiment, the dose will be from about 10 milligrams to 450 milligrams. In another embodiment, the dose will be from about 25 to about 250 milligrams. In another embodiment, the dose will be about 50 or 100 milligrams. In one embodiment, the dose will be approximately 100 milligrams. It is considered that the compound can be administered о once, twice or thrice a day.

Although it is possible that the active ingredients are administered alone, they may preferably be presented as pharmaceutical compositions. The compositions, for both veterinary and human use, according to the invention comprise at least one active ingredient, as defined above, together with one or more carriers acceptable for this and, optionally, other therapeutic ingredients. The carrier (s) must be "acceptable" in the sense of being compatible with the other ingredients in the composition and physiologically harmless to the recipient.

MD 4403 Cl 2016.09.30

The compositions include those suitable for the administration methods mentioned above. The compositions may conveniently be presented in single dosage form and may be prepared by any of the methods well known in the field of pharmacy. Methods and compositions are generally found in [29]. Such methods include the step of bringing the active ingredient together with the carrier constituting one or more auxiliary ingredients. Generally, the compositions are prepared by bringing the active ingredient in uniform and intimate association with the liquid carriers or the finely divided solid carriers or both, and then, if necessary, forming the product.

The compositions of the present invention suitable for oral administration may be presented in the form of discrete units such as capsules, boxes or tablets, each containing a predetermined amount of active ingredient; in the form of powder or granules; as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient can also be administered as a bowl, electro or paste.

The tablet is prepared by compression or tuming, optionally with one or more auxiliary ingredients. The compressed tablets can be prepared by compressing in a suitable machine the active ingredient in a free flowing form, such as о powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surfactant or dispersing agent. . The poured tablets can be manufactured by pouring them into a suitable machine of a powdered active ingredient mixture, moistened with an inert liquid diluent. The tablets can be optionally coated or labeled and optionally formulated to provide controlled release of the active ingredient from them.

For administration to the eyes or other extraneous tissues, for example, mouth and skin, the compositions are preferably applied as an ointment or cream containing the active ingredient (s) in an amount of, for example, 0.075 to 20% w / w (including active ingredient (s) in a range of 0.1% to 20% in steps of 0.1% w / w, such as 0.6% w / w, 0.7% g (g, etc.), preferably from 0.2 to 15% w / w and most preferably from 0.5 to 10% w / w. When formulated in an ointment, the active ingredients can be used with either a paraffinic or a water miscible ointment base. Alternatively, the active ingredients may be formulated in an oil-in-water-based cream.

If desired, the aqueous phase of the cream base may include, for example, at least 30% w / w of polyhydroxyl alcohol, i.e. an alcohol having two or more hydroxyl groups, such as propylene glycol, butan 1,3-diol , mannitol, sorbitol, glycerol and polyethylene glycol (including PEG 400) and mixtures thereof. Topical compositions may preferably comprise a compound that increases the absorption or penetration of the active ingredient through the skin or other affected areas. Examples of such dermal penetration enhancers include dimethylsulfoxide and related analogues.

The oil phase of the emulsions of this invention may consist of known ingredients in a known manner. While the phase may comprise only an emulsifier (otherwise known as an emulsifier), it preferably comprises a mixture of at least one emulsifier with о fat or an oil or with both о fat and an oil. Preferably, a hydrophilic emulsifier is included together with a lipophilic emulsifier that acts as a stabilizer. It is also preferable to include both oil and fat. Together, the emulsifier (s) with or without stabilizer (s) form the so-called emulsifying wax, and the wax along with the oil and fat forms the so-called emulsifying ointment that forms the oil-dispersed phase of the cream compositions.

Emulsifiers and emulsion stabilizers suitable for use in the composition of the invention include Tween® 60, Span® 80, cetostearyl alcohol, benzyl alcohol, myristyl alcohol, glyceryl mono-stearate and sodium lauryl sulfate.

Choosing the right oils or fats for the composition is based on achieving the desired cosmetic properties. The cream should preferably be a non-stainy, non-stainable, washable product with adequate consistency to prevent leakage from tubes or other containers. Mono- or dibasic alkyl esters with straight or branched chains, such as di-isoadipate, isocetyl stearate, propylene glycol, coconut fatty acid diester, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2- palmitate ethylhexyl or a mixture of branched chain esters known as Crodamol CAP can be used, the last three being preferred esters. They can be used alone or in combination depending on the required properties. Alternatively, yes

MD 4403 Cl 2016.09.30 use high melting point lipids such as soft white and / or liquid paraffin or other mineral oils.

The pharmaceutical compositions of the present invention contain one or more compounds of the invention together with one or more pharmaceutically acceptable carriers or excipients and optionally other therapeutic agents. Pharmaceutical compositions containing the active ingredient may be in any form suitable for the method for administration. When used for oral use, for example, tablets, tablets, pills, aqueous or oily suspensions, dispersible granules or powders, emulsions, soft or hard capsules, syrups or elixirs may be prepared. Compositions intended for oral use may be prepared according to any method known in the art for the manufacture of pharmaceutical compositions and such compositions may confine one or more agents, including sweeteners, flavoring agents, coloring agents and preservatives, for the purpose. to provide an acceptable preparation. Tablets containing the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients, which are suitable for tablet manufacture, are acceptable. These excipients may be, for example, inert diluents, such as calcium or sodium carbonate, lactose, lactose monohydrate, croscarmellose sodium, povidone, calcium or sodium phosphate; granulating and disintegrating agents, such as corn starch or alginic acid; lymphomas such as cellulose, microcrystalline cellulose, starch, gelatin or acacia; and lubricating agents, such as magnesium stearate, stearic acid or talc. Tablets may be uncoated or coated by known techniques, including microcapsulation to refine disintegration and adsorption into the gastrointestinal tract and thereby provide lasting action for a longer period. For example, a time delay material, such as glyceryl monostearate or glyceryl distearate alone or with wax, may be used.

Oral compositions may also be presented as gelatin capsules if the active ingredient is mixed with a solid inert diluent, for example, calcium phosphate or kaolin, or as soft gelatin capsules, wherein the active ingredient is mixed with water or an oil medium, such as peanut oil, liquid paraffin or olive oil.

The aqueous suspensions of the invention contain the active substances mixed with suitable excipients for the manufacture of aqueous suspensions. Such excipients include a suspending agent, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose, sodium alginate, polyvinylpyrrolidone, tragacanth gum and arabic gum, and dispersing or wetting agents, such as natural phosphate, e.g. ), a condensation product of an alkylene oxide with a fatty acid (eg, polyoxyethylene stearate), a condensation product of ethylene oxide, with a long cycle of aliphatic alcohol (e.g., heptadecaetylene glycol ethanol), a condensation product of ethylene oxide with an ester partially derived from fatty acid and о anhydride hexitol (for example polyoxyethylene sorbitan monooleate). The aqueous suspension may also contain one or more preservatives, such as ethyl or n-propyl p-hydroxy-benzoate, one or more coloring agents, one or more flavoring agents, and one or more flavoring agents. sweetening, such as sucrose or saccharin.

Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, such as peanut oil, olive oil, coconut oil or sesame oil, or in a mineral oil, such as liquid paraffin. Oral suspensions may contain a thickening agent, such as beeswax, heavy paraffin or cetyl alcohol. Sweetening agents, such as those presented above, and flavoring agents may be added to obtain an acceptable oral preparation. These compounds can be preserved by the addition of an antioxidant such as ascorbic acid.

The dispersible powders and granules of the invention suitable for the preparation of an aqueous suspension by the addition of water produce an active ingredient mixed with a dispersing or wetting agent, a suspending agent as well as one or more preservative sulfates. Suitable dispersing or wetting agents and suspending agents are exemplified by those described above. Additional excipients, for example, sweetening, flavoring and coloring agents may also be present.

The pharmaceutical compositions of the invention may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil, such as olive oil or peanut oil, a mineral oil, such as liquid paraffin, or a mixture thereof.

MD 4403 Cl 2016.09.30

Suitable emulsifying agents include natural gums such as gum arabic and tragacanth gum, naturally occurring phosphatides such as soy lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides such as sorbitan monooleate and condensation products of these esters partially! with ethylene oxide, such as polyoxyethylene sorbitan monooleate. The emulsion can also contain sweetening agents and flavorings. The syrups and elixirs can be formulated with sweetening agents such as glycerin, sorbitol or sucrose. Such compositions may also contain a soothing, preservative, flavoring or coloring agent.

The pharmaceutical compositions of the invention may be in the form of a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated according to the prior art known using suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as a solution in 1,3-butan-diol or a lyophilized powder preparation. Acceptable fillers and solvents that can be used include ара, Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils can be conventionally used as a solvent or suspending medium. For this purpose, any non-volatile sedative oil can be used, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid may also be used in the preparation of injectable products.

The amount of active ingredient that can be combined with carrier materials to produce a single dosage form will vary depending on the treated host and the particular mode of administration. For example, a prolonged-acting composition intended for oral administration in humans may contain about 1 to 1000 mg of active material combined with a suitable and convenient amount of carrier material, which may vary from about 5 to about 95% of the total composition ( weight: weight). The pharmaceutical composition can be prepared to obtain easily measurable quantities for administration. For example, an aqueous solution intended for intravenous infusion may contain from about 3 to 500 µg of active ingredient per milliliter of solution so that the infusion can be produced with an appropriate volume at a flow rate of about 30 mL / hour.

Suitable compositions for administration to the eye include eye drops in which the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent for the active ingredient. The active ingredient is preferably present in such compositions in a concentration of 0.5 to 20%, advantageously 0.5 to 10%, especially about 1.5% w / w.

Suitable compositions for topical administration to the mouth include tablets containing the active ingredient in a flavored base, usually sucrose and acacia or tragacanth; pills containing the active ingredient in an inert base, such as gelatin and glycerine, or sucrose and acacia; and mouthwash solutions containing the active ingredient in a suitable liquid carrier.

The compositions for rectal administration may be presented as a suppository with a suitable base containing, for example, cocoa butter or salicylate.

Compositions suitable for nasal or intrapulmonary administration have о particle size, for example, in the range of 0.1 to 500 microns (including particle sizes in the range of 0.1 to 500 microns in microns, such as 0, 5, 1, 30 microns, 35 microns, etc.), which are administered by rapid inhalation through the nasal meatus or by inhalation through the mouth so as to reach the alveolar sacs. Suitable compositions include aqueous or oily solutions of the active ingredient. Compositions suitable for the administration of dry powder or aerosol can be prepared according to conventional methods and may be delivered with other therapeutic agents, such as compounds used so far in the treatment or prophylaxis of conditions associated with HCV activity.

Compositions suitable for vaginal administration may be presented as ova, swabs, creams, gels, pastes, foams or spray compositions which further confine the active ingredient to such carriers as those known in the art as suitable.

Suitable compositions for parenteral administration include sterile injectable aqueous and non-aqueous solutions that may contain antioxidants, buffers, bacteriostats and

MD 4403 Cl 2016.09.30 solufs making the isotonic composition with the blood of the treated container; and sterile aqueous and non-aqueous suspensions which may include suspending agents and thickening agents.

The compositions are presented in single-dose or multi-dose containers, for example sealed vials and vials, and can be stored in a lyophilized state that only requires the addition of a sterile liquid carrier, such as water for injection, immediately prior to use. Extemporaneous injectable solutions and suspensions are prepared from sterile powders, granules and tablets of the type described above. Preferred unit dose compositions are those which contain a daily dose or daily unit sub-dose, as mentioned above, or a suitable fraction thereof, of active ingredient.

It should be understood that, in addition to the ingredients mentioned above, the compositions of this invention may include other conventional agents! In the art, taking into account the type of composition in question, for example, those suitable for oral administration may include flavoring agents.

The invention further provides veterinary compositions containing at least one active ingredient as defined above, together with a veterinary carrier.

Veterinary carriers are useful materials for the purpose of administering the composition and may be solid, liquid or gaseous materials that are otherwise inert or acceptable in the veterinary field and are compatible with the active ingredient. These veterinary compounds may be administered orally, parenterally or by any other desired route.

The compounds of the invention may also be formulated to provide controlled release of the active ingredient, to allow more frequent dosing or to improve the pharmacokinetic profile or toxicity of the active ingredient. Accordingly, the invention also provides for compositions containing one or more compounds of the invention formulated for sustainable or controlled ease.

The effective dose of the active ingredient depends on the nature of the disorder, the toxicity, whether the compound is used prophylactically (lower doses), the method of administration, as well as the pharmaceutical composition, and will be determined by the physician using conventional dose escalation studies. .

Routes of administration

One or more compounds of the invention (hereinafter referred to as active ingredients) are administered by any means appropriate to the condition being treated. Suitable pathways include oral, rectal, nasal, topical (including oral and sublingual), vaginal and parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural) and the like. It will be determined that the preferred path may vary, for example, with the condition of the container. An advantage of the compounds of the invention is that they are bioavailable orally and can be dosed orally.

HCV-associated therapy

In another embodiment, non-limiting examples of suitable combinations include combinations of one or more compounds of general formula (I) and (A1-A4), with one or more interferons, ribavirin or its analogues, HCV protease inhibitors. NS3, alpha-glucosidase 1 inhibitors, hepatoprotectants, nucleoside or nucleotide inhibitors of HC5 NS5B polymerase, non-nucleoside inhibitors of VHC NS5B polymerase, HCV inhibitors NS5A, TLR-7 agonists, cyclophilin inhibitors, HCV IRES inhibitors, pharmacokinetic enhancers and other drugs or therapeutic agents for treating HCV.

More precisely, one or more compounds, as described herein, may be combined with one or more compounds selected from the group consisting of:

1) interferons, for example, pegylated rIFN-alpha 2b (PEG-INTRON), pegylated rIFN-alpha 2a (Pegasys), rIFN-alpha 2b (Intron A), rIFN-alpha 2a (Roferon-A), interferon alpha (MOR) -22, OPC-18, Alpha, Alfanative, Multifer, subalin), interferon alpha-1 (Infergen), interferon alpha-nl (Wellferon), interferon alpha-n3 (Alferon), interferon-beta (Avonex, DL-8234) , interferon-omega (omega DUROS, Biomed 510), albinterferon alpha-2b (Albuferon), IFN alpha-2b XL, BLX-883 (Locteron), DA-3021, glycosylated interferon alpha-2b (AVI-005), PEG-Infergen, interferon lambda-1 (PEGylated IL-29) and belerophon;

2) ribavirin and its analogues, for example, ribavirin (Rebetol, Copegus) and taribavirin (Viramidine)

3) VHC NS3 protease inhibitors, for example, boceprevir (SCH-503034, SCH-7), telaprevir (VX-950), TMC435350, BI-1335, BI-1230, MK-7009, VBY-376, VX-500 ,

MD 4403 Cl 2016.09.30

GS -9256, GS-9451, BMS-605339, PHX-1766, AS-101, YH-5258, YH5530, YH5531, ABT-450, ACH-1625, ITMN-191, MK5172, MK6325 and MK2748;

4) alpha-glucosidase 1 inhibitors, for example, celgosivir (MX-3253), miglitol and UT-231B;

5) hepatoprotectants, for example, emericasan (IND-6556), ME-3738, GS-9450 (LB84451), silibilin and MitoQ;

6) nucleoside or nucleotide inhibitors of HCV NS5B polymerase, for example, R1626, R7128 (R4048), IDX184, IDX-102, BCX-4678, valopicitabine (NM-283), MK0608, sofosbuvir (GS-7977 (formerly PSI-7977) )) and INX-189 (now BMS986094);

7) non-nucleoside inhibitors of HC5 NS5B polymerase, for example, PF-868554, VCH-759, VCH-916, JTK-652, MK-3281, GS-9190, VBY-708, VCH-222, A848837, ANA- 598, GL60667, GL59728, A-63890, A-48773, A-48547, BC-2329, VCH-796 (nesbuvir), GSK625433, BILN-1941, XTL-2125, ABT-072, ABT-333, GS-9669 , PSI7792 and GS-9190;

8) HC5 NS5A inhibitors, for example, AZD-2836 (A-831), BMS-790052, ACH3102, ACH-2928, MK8325, MK4882, MK8742, PSI-461, IDX719 and A-689;

9) TLR-7 agonists, for example, imichimod, 852A, GS-9524, ANA-773, ANA-975, AZD-8848 (DSP-3025) and MS, 360,320;

10) cyclophilin inhibitors, for example, Debio-025, SCY-635 and NIM811;

11) IRES RNA inhibitors, for example, MCI-067;

12) pharmacokinetic amplifiers, for example, BAS-100, SPI-452, PF-4194477, TMC-41629, GS-9350, GS-9585 and roxithromycin; and

13), other medicines for treating HCV, for example, alpha thymosin 1 (Zadaxin), nitazoxanide (Alinea, NTZ), BIVN-401 (virostat), PYN-17 (altirex), KPE02003002, actilon (CPG-10101), GS -9525, KRN-7000, civacir, GI-5005, XTL-6865, BIT225, PTX-111, ITX2865, TT-033i, ANA 971, NOV-205, tarvacine, EHC-18, VGX-410C, EMZ-702, AVI 4065, BMS-650032, BMS-791325, Bavituximab, MDX-1106 (ONO4538), Oglufanide and VX-497 (merimepodib).

More specifically, one or more compounds, as described herein, may be combined with one or more compounds selected from the group consisting of non-nucleoside HCV NS5B polymerase inhibitors (ABT-072 and ABT-333), inhibitors of HCV NS5A (ACH-3102 and ACH-2928) and HCV NS3 protease inhibitors (ABT-450 and ACH-1625).

In another embodiment, the present application discloses pharmaceutical compositions containing a compound as described herein, or a pharmaceutically acceptable salt, solvate, and / or ester thereof, in combination with at least one additional therapeutic agent, and a pharmaceutically acceptable carrier or excipient.

According to an example, the therapeutic agent used in combination with the compound, as described herein, may be any agent having a therapeutic effect when used in combination with the compound described herein. For example, the therapeutic agent used in combination with the compound described herein may be interferons, ribavirin analogues, NS3 protease inhibitors, NS5B polymerase inhibitors, alfaglucosidase 1 inhibitors, hepatoprotectants, non-nucleoside HCV inhibitors and other HCV treatment drugs.

In another embodiment, the present application provides pharmaceutical compositions containing a compound of general formula (I), or a pharmaceutically acceptable salt, a solvate and / or an ester thereof, in combination with at least one additional therapeutic agent selected from group consisting of pegylated rIFN-alpha 2b, pegylated rIFN-alpha 2a, rIFN-alpha 2b, IFN alpha-2b XL, rIFN-alpha 2a, IFN alpha consensus, inferential, rebif, locteron, AVI-005, PEG-inferential, IFN - pegylated beta, oral alpha interferon, FERON, referon, INTERMAX alpha, r-IFN-beta, infergen + actimun, IFN-omega with DUROS, albuferon, rebetol, copegus, levovirine, VX-497, viramidine (taribavirin), A-831, A-689, NM283, valopicitabine, R1626, PSI-6130 (R1656), VHC-796, BILB 1941, MK-0608, NM107, R7128, VCH-759, PF-868554, GSK625433, XTL-2125, SCH-503034 (SCH-7), VX-950 (Telaprevir), ITMN-191 and BILN-2065, MX-3253 (celgosivir), UT-231B, IDN6556, ME 3738, MitoQ and LB-84451, benzimidazole derivatives, benzo derivatives

1,2,4-thiadiazine and phenylalanine derivatives, zadaxine, nitazoxanide (alignment), BIVN-401 (virostat), Debio-025, VGX-410C, EMZ-702, AVI 4065, bavituximab, oglufanide, PYN-17, KPE02003 , actilon (CPG-10101), KRN-7000, civacir, GI-5005, ANA-

MD 4403 Cl 2016.09.30

975 (izatoribine), XTL-6865, ANA 971, NOV-205, tarvacin, EHC-18 and NIM81I, and a pharmaceutically acceptable carrier or excipient.

In another embodiment, the present application provides a combination pharmaceutical agent comprising:

a) о first pharmaceutical composition containing a compound of general formula (I), or о a pharmaceutically acceptable salt, solvate or ester thereof, and

b) о the second pharmaceutical composition containing at least one additional therapeutic agent selected from the group consisting of inhibitory compounds! you have HIV protease, inhibitor! non-nucleoside HIV tr a reverse aze script, inhibitor! HIV nucleoside tr inv script, reverse inhibitor! nucleotides of HIV reverse transcriptase inhibitor! you have HIV integrase, inhibitor! you have gp41, inhibitor! you have CXCR4, inhibitor! you have gpl20, inhibitor! to CCR5, interferons, analogues to ribavirin, inhibitive! of NS3 protease, inhibitor! you have alfaglucosidase 1, hepatoprotectants, inhibitor! non-nucleoside HCV and other medicines for treating HCV, and combinations thereof.

In another embodiment, a pharmaceutical composition containing a compound of general formula (I), as described herein and a nucleoside or nucleotide inhibitor of HCV NS5B polymerase and optionally an interferon or ribavirin, is described. In one embodiment, the compound represents methyl {(2S) -l - [(2S, 5S) -2- (9- {2 - [(2S, 48) -1 {(2R) -2- [(methoxycarbonyl ) amino] -2-phenylacetyl} -4- (methoxymethyl) pyrrolidine-2-yl] -1Himidazol-5-yl) -1,11-dihydroisochromeno [4 ', 3': 6,7] naphtha [1,2- d] imidazol-2-yl) -5-methylpyrrolidine-1-yl] -3-methyl-1-oxobutan-2-yl) carbamate of the formula:

<img file="MD4403C1_D0003.tif" />

О and the inhibitor is sofosbuvir.

Combinations of compounds of formula I and adiphonal active therapeutic agents may be selected for the treatment of HCV-infected patients and other conditions such as HIV infections. Accordingly, compounds of formula I may be combined with one or more compounds useful in the treatment of HIV, for example inhibitory compounds! you have HIV protease, inhibitor! non-nucleoside HIV reverse transcriptase inhibitor! HIV nucleoside reverse transcriptase inhibitor! nucleotides of HIV reverse transcriptase inhibitor! you have HIV integrase, inhibitor! you have gp41, inhibitor! you have CXCR4, inhibitor! to gpl20, CCR5 inhibitors, interferons, ribavirin analogues, NS3 protease inhibitors, NS5b polymerase inhibitors, alpha-glucosidase 1 inhibitors, hepatoprotectants, non-nucleoside HCV inhibitors and other HCV treatment drugs

Specifically, one or more compounds of general formula (I) may be combined with one or more compounds selected from the group consisting of 1) HIV protease inhibitors, for example, amprenavir, atazanavir, fosamprenavir, indinavir, lopinavir, ritonavir, lopinavir + ritonavir, nelfinavir, sachinavir, tipranavir, brecanavir, darunavir, TMC-126, TMC-114, mozenavir (DMP-450), JE-2147 (AG1776), AG1859, DG35, L756423, RO0334649, KNI-272, DPC 681, DPC-684 and GW640385X, DG17, PPL-100, 2), a non-nucleoside reverse HIV transcriptase inhibitor, for example, capravirine, emivirine, delaviridine, efavirenz, nevirapine, (+) calanolid A, etravirine, GW5634, DPC-083, DPC-961, DPC-963, MIV-150, and TMC-120, TMC-278 (rilpivirine), efavirenz, BILR 355 BS, VRX 840,773, UK-453,061, RDEA806; 3) a nucleoside reverse transcriptase inhibitor of HIV, for example, zidovudine, emtricitabine, didanosine, stavudine, zalcitabine, lamivudine, abacavir, amdoxovir, elvucitabine, allovudine, MIV-210, racivir (+ -FTC), D-dabin, fosfazide, fosoxudud tidoxil, tidoxil fosalvudine, apricitibine (AVX754), amdoxovir, KP-1461, abacavir + lamivudine, abacavir + lamivudine + zidovudine, zidovudine + lamivudine, 4), a nucleotide reverse transcriptase inhibitor, HIV reverse transcriptase inhibitor tenofovir, tenofovir disoproxil + emtricitabine fumarate, tenofovir disoproxil + emtricitabine + efavirenz, and

MD 4403 Cl 2016.09.30 adefovir, 5) an inhibitor of HIV integrase, for example, curcumin, curcumin derivatives, chicoric acid, chicoric acid derivatives, 3,5-dicaffeoilquinic acid, 3,5-dicaffeoilquinic acid derivatives, aurintricarboxylic acid, aurintricarboxylic acid derivatives, caffeic acid phenethyl ester, caffeic acid phenethyl ester derivatives, typhostin, typhostin derivatives, keratin, keratin derivatives, S-1360, zintevir (AR-177), L-870812 and L- 870810, MK-0518 (raltegravir), BMS-707035, MK2048, BA-011, BMS-538158, GSK364735C, 6), a gp41 inhibitor, e.g., enfuvirtide, sifuvirtide, FB006M, TRI- 1144, SPC3, DES6, Locus gp41, CovX and REP 9, 7) a CXCR4 inhibitor, for example, AMD-070, 8) an input inhibitor, for example, SP01A, TNX-355, 9) a gpl20 inhibitor , for example, BMS-488043 and BlockAide / CR, 10) an inhibitor of G6PD and NADH-oxidase, for example, immunitin, 10) an inhibitor of CCR5, e.g. aplaviroc, vicriviroc, INCB9471, PRO-140, INCB15050, PF-232798, CCR5mAb004 and maraviroc, 11) an interferon, for example, pegylated rIFN-alpha 2b, pegylated rIFN-alpha 2, rIFN-alpha 2b, IFN alpha-2b XL , rIFN-alpha 2a, consensus IFN alpha, infergene, Rebif, locteron, AVI-005, PEG-nurse, IFN-beta pegylated, alpha oral interferon, iron, reaferon, lintermax alpha, r-IFN-beta, infergen + actimun, IFNomega with DUROS and albuferon, 12) ribavirin analogues, for example, rebetol, copegus, levovirin, VX-497 and viramidine (taribavirin) 13) NS5A inhibitors, e.g., A831, A-689 and BMS-790052, 14), NS5B polymerase inhibitors, for example, NM-283, valopicitabine, R1626, PSI-6130 ( R1656), VHC-796, BILB 1941, MK-0608, NM-I07, R7128, VHC-759, PF-868554, GSK625433 and XTL-2125, 15), NS3 protease inhibitors, e.g., SCH-503034 (SCH -7), VX-950 (Telaprevir), ITMN-191 and BILN-2065, 16), alpha-glucosidase 1 inhibitors, for example, MX-3253 (celgosivir) and UT-231B, 17) hepatoprotectants, for example, IND-6556, ME 3738, MitoQ and LB84451, 18), non-nucleoside HCV inhibitors, for example, benzimidazole derivatives, benzo-1, 2,4-thiadiazine derivatives and phenylalanine derivatives , 19) other medicines for treating hepatitis C, for example, zadaxin, nitazoxanide (alignment), BIVN -401 (virostat), Debio-025, VGX-410C, EMZ-702, AVI 4065, bavituximab, oglufanide, PYN-17, KPE02003002, actilon (CPG-10I0I), KRN-7000, civacir, GI5005, ANA-975 (izatoribine), XTL-6865, ANA 971, NOV-205, tarvacin, EHC-18 and NIM811, 19) pharmacokinetic enhancers, for example, BAS-100 and SPI452, 20) H RNA -ase inhibitors, for example, ODN-93 and ODN -112, 21), other anti-HIV drugs, for example, VGV-1, PA-457 (bevirimate), amplifier, HRG214, cytolin, polymon, VGX-4I0, KD247, AMZ 0026, CYT 99007, HIV A-221, BAY 50-4798, MDX010 (iplimumab), PBS119, ALG889 and PA-1050040.

0116 It is considered that the second therapeutic agent will be administered in a manner known in the art, and the dose may be selected by one skilled in the art. For example, the second agent may be administered in a dose of from about 0.01 milligrams to about 2 grams per day.

Metabolites of compounds

Also, the in vivo metabolic products of the compounds described in the present invention are included in the scope of this invention. Such products may result, for example, from oxidation, reduction, hydrolysis, amidation, esterification and the like of the administered compound, primarily due to enzymatic processes. Accordingly, the invention includes compounds produced by a process which includes contacting a compound of the invention with a mammal for a sufficient period of time to produce a metabolic product thereof. Such products are usually identified by the preparation of a radioactive labeled compound (for example, C.¹⁴ or H³) of the invention, its parenteral administration at a detectable dose (eg, greater than about 0.5 mg / kg) in an animal, such as rat, mouse, guinea pig, mackerel or human, allowing sufficient time to produce metabolism (usually about 30 seconds to 30 hours) and to isolate conversion products from urine, blood or other biological samples. These products are easy to isolate because they are labeled (others are isolated by the use of antibodies capable of binding the epitopes that survive in the metabolite). The metabolite structures are conventionally determined, for example, by MS or NMR analysis. In general, metabolism analysis is done in the same way as conventional drug metabolism studies well known to those working in the art. Conversion products, as long as they are not otherwise found in vivo, are useful in diagnostic tests

MD 4403 Cl 2016.09.30 for the therapeutic dosage of the compounds of the invention even though they do not possess inhibitory activity of their own HCV.

Methods for determining the stability of compounds in surrogate gastrointestinal secretions are known.

Exemplary methods for the preparation of compounds

The invention also relates to methods for preparing the compositions of the present invention. The compositions are prepared by any of the applicable methods of organic synthesis. Many such methods are well known in the art. However, many of the known methods are developed in the Compendium of Organic Synthetic Methods [30], [31], [32], [33], [34], [35], and [36], [37]. Other suitable methods for the preparation of the compounds of the invention are described in the international patent application [38]. ''

A number of exemplary methods for preparing the compositions of the invention are provided in the schemes and examples below. These methods are intended to illustrate the nature of such preparations and do not intend to limit the scope of the applicable methods.

Generally, the reaction conditions, such as temperature, reaction time, solvents, treatment procedures and the like, will be those common in the art for performing the special reaction. The cited reference material, together with the material cited here, contains detailed descriptions of these conditions. Usually the temperatures will be from 100 ° C to 200 ° C, the solvents will be aprotic or protic, and the reaction times will be from 10 seconds to 10 days. Treatment usually includes quenching any unreacted reagents followed by separation between an organic water / layer system (extraction) and separation of the boundary layer of the product.

Oxidation and reduction of reactions are usually carried out at temperatures close to room temperature (approximately 20 ° C), although for metal hydride reduction the temperature is frequently reduced to 0 ° C to -100 ° C, solvents are usually suitable for reductions and can be either protic or aprotic for oxidation. The reaction times are adjusted to achieve the desired conversions.

Condensation reactions are usually performed at temperatures close to room temperature, although for non-equilibrium, the reduced temperatures (from 0 ° C to 100 ° C) of the kinetically controlled condensations are also common. Solvents can be protic (common in reaction balancing) or aprotic (common in kinetically controlled reactions).

Standard synthetic methods, such as azeotropic removal of reaction products and use of anhydrous reaction conditions (for example, inert gas media) are common in the field and are applied where appropriate.

The terms "treated", "treated", "treated" and the like, when used in connection with a synthetic synthetic operation, means contact, mixing, reacting, permitting to react, contact, and other common terms in the art. to indicate that one or more chemical entities are treated in such a way that they are converted to one or more other chemical entities. This means that "treating a compound with compound two" is synonymous with "allowing compound one to react with compound two", "contacting compound one with compound two", "reaction of compound one with compound two" and other common expressions in the art. organic synthesis to reasonably indicate that compound one was "treated", "reacted", "allowed to react", etc., with compound two. For example, the treatment indicates the reasonable and usual way in which organic chemical substances are allowed to react. Normal concentrations (from 0.01M to 10M, accurate from 0.1M to IM), temperatures (from -100 ° C to 250 ° C, accurate from -78 ° C to 150 ° C , more precisely from

-78 ° C to 100 ° C, more precisely 0 ° C to 100 ° C), reaction vessels (usually glass, plastic, metal), solvents, pressures, atmospheres (usually air for insensitive reactions with oxygen and water or nitrogen or argon for oxygen or water sensitive), etc., are intended unless otherwise indicated. Knowledges of similar reactions, known in the field of organic synthesis, are used in the selection of conditions and devices for "treatment" in a given process. In particular, a specialist in the field of organic synthesis selects the conditions and apparatus in a reasonable manner in order to successfully carry out the chemical reactions from the processes described based on the knowledge in the art.

MD 4403 Cl 2016.09.30

Modifications of each of the exemplary schemes and in the Examples (hereinafter referred to as "exemplary schemes") lead to different analogues of the specific exemplary materials. The above quotes describing the appropriate methods of organic synthesis apply for such modifications.

In each of the exemplary schemes it may be advantageous to separate the reaction products from one another and / or from the initiated materials. The desired products of each step or series of steps are separated and / or purified (further separated) to the desired degree of homogeneity by the common methods in the art. Typically, these separations involve multi-phase extraction, crystallization of a solvent or solvent mixture, distillation, sublimation or chromatography. Chromatography can involve any number of methods, including, for example: reverse phase and normal phase; steric exclusion, ion exchange; high, medium and low pressure; methods and apparatus for liquid chromatography; small-scale analytics; simulated mobile bed (PMS) and preparative thin or thick layer chromatography, as well as small scale rapid chromatography techniques.

Another class of separation methods involves treating a mixture with a reagent of choice to bind to or otherwise separate a desired product, an unreacted starting material, reaction by product or the like. Such reagents include adsorbents or absorbers, such as activated carbon, molecular sites, ion exchange media or the like. Alternatively, the reagents may be acidic in the case of a base material, bases in the case of an acidic material, binding reagents such as antibodies, binding proteins, selective chelators such as macrocyclic ethers, liquid / liquid extraction reagents. (LIX) or the like.

The selection of appropriate separation methods depends on the nature of the materials involved. For example, the boiling point and the molecular weight in distillation and sublimation, the presence or absence of polar functional groups in chromatography, the stability of materials in acidic and basic environments in multiphase extraction and the like. One skilled in the art will most likely apply techniques to achieve the desired separation.

A single stereoisomer, for example, an enantiomer, substantially free of its stereoisomer, can be obtained by resolving the racemic mixture using a method such as diastereomeric formation using optically active resolving agents [39]. Racemic mixtures of the chiral compounds of the invention may be separated and isolated by any suitable method, including: (1) formation of ionic diastereomeric salts with chiral compounds and separation by fractional crystallization or other methods, (2) formation of diastereomeric compounds with chiral derivatization reagents, separation of diastereomers and conversion into pure stereoisomers, and (3) separation of pure or enriched stereoisomers directly into chiral condices.

According to method (1), diastereomeric salts can be formed by reacting chiral enantiomerically pure bases such as brucine, quinine, ephedrine, strychnine, cc-methyl-β-phenylethylamine (amphetamine) and the like, with asymmetric compounds having acid functionality, such as acid carboxylic acid and sulfonic acid. Diastereomeric salts can be induced to separate by fractional crystallization or ion chromatography. For the separation of the optical isomers of amino compounds, the addition of chiral carboxylic or sulfonic acids, such as camphorsulfonic acid, tartaric acid, mandelic acid or lactic acid can lead to the formation of diastereomeric salts.

Alternatively, by method (2), the substrate to be resolved is reacted with an enantiomer of a chiral compound to form a diastereomeric pair [40]. Diastereomeric compounds can be formed by reacting asymmetric compounds with chiral enantiomerically pure derivatization reagents, such as mentile derivatives, followed by separation of diastereomers and hydrolysis to obtain an enantiomerically enriched free substrate. The method of determining optical purity involves the production of chiral esters, such as a methyl ester, for example, (-) chloroformate menthol in the presence of a base, or a Mosher ester, cc-methoxy-a- (trifluoromethyl) phenyl acetate [ 41], of the racemic mixture, and the NMR spectrum analysis for the presence of the two atropisomeric diastereomers. The stable diastereomers of the atropisomeric compounds can be separated and isolated by reverse and normal phase chromatography based on atropisomeric naphthyl isoquinoline separation methods [42]. By method (3), a racemic mixture of two enantiomers can be separated by chromatography using the chiral stafion phase [43]. Enriched or purified enantiomers can be distinguished by the methods used to distinguish others

MD 4403 Cl 2016.09.30 chiral molecules with asymmetric carbon atoms, such as optical rotation and circular dichroism.

Schemes and examples

The general aspects of these exemplary methods are described below in the Examples. Each of the products in the following processes is optionally separated, isolated and / or purified before use in subsequent processes.

A number of exemplary methods for preparing compounds of the invention is provided herein, for example, in the examples below. These methods are intended to illustrate the nature of such preparations and are not intended to limit the scope of the applicable methods. Certain compounds of the invention may be used as intermediates for the preparation of other compounds of the invention. In the exemplary methods described herein, the EV- fragment may also be written as R9-. PG represents о common protective group for the given functional group that is attached. The installation and removal of the protective assembly can be performed using standard methods, such as those described in [44].

Scheme 1. Representative synthesis of EVC (= O) -PWPC (= O) -VE

H₂NVC (= O) -PWPC (= O) -ve

1

<img file="MD4403C1_D0004.tif" />

H₂NVC (= O) -PWPC (= O) -V-NH₂

<img file="MD4403C1_D0005.tif" />

NH-VC (= O) -PWPC (= O) -VE ° \ ^1b

О c

NH-VC (= O) -PWPC (= O) -VN H—

Scheme 1 shows a general synthesis of a molecule of the invention EVC (= O) -P-WP-C (= O) -VE in which, for illustrative purposes, E represents methoxycarbonylamino. By treating either α or 1c with one or two equivalents of methyl chloroformate respectively under basic conditions (for example, sodium hydroxide), molecule I or II is obtained.

Scheme 2. Representative synthesis of EVC (= O) -PWPC (= O) -VE

<img file="MD4403C1_D0006.tif" />

2 p.m.

HO ^ SEE

О

2b

<img file="MD4403C1_D0007.tif" />

2c

<img file="MD4403C1_D0008.tif" />

<img file="MD4403C1_D0009.tif" />

HO ^ SEE

О

2b

<img file="MD4403C1_D0010.tif" />

Scheme 2 shows a general synthesis of a molecule of the invention EVC (= O) -P-WP-C (= O) -VE in which, for illustrative purposes, P represents pyrrolidine. The coupling of the amine 2a with the acid 2b is carried out using a peptide coupling agent (eg HATU) to obtain 2c. Alternatively, amine 2d is coupled with two equivalents of 2b under similar conditions to obtain 2e.

MD 4403 Cl 2016.09.30

Scheme 6. Representative synthesis of R ^ VC ^ Oj-PR²

<img file="MD4403C1_D0011.tif" />

ба H

HO ^ —V-NH-PG

О

6b

<img file="MD4403C1_D0012.tif" />

<img file="MD4403C1_D0013.tif" />

HO

О

6e

PG-HN-VC (= O) -PW

<img file="MD4403C1_D0014.tif" />

<img file="MD4403C1_D0015.tif" />

PG-HN-VC (= O) -PW

<img file="MD4403C1_D0016.tif" />

HO + V-NH-PG

О

6b

PG-HN-VC (= O) -PW e.g.

<img file="MD4403C1_D0017.tif" />

V-NH-PG

6d

<img file="MD4403C1_D0018.tif" />

HO

VE

6e

<img file="MD4403C1_D0019.tif" />

<img file="MD4403C1_D0020.tif" />

PG-HN-PW

PG-HN-PW

<img file="MD4403C1_D0021.tif" />

HO

VV-NH-PG

О

6b

<img file="MD4403C1_D0022.tif" />

<img file="MD4403C1_D0023.tif" />

V-NH-PG

PG-HN-W

<img file="MD4403C1_D0024.tif" />

HO ^ SEE

О

6e

<img file="MD4403C1_D0025.tif" />

PG-HN-W

PG-HN-W

<img file="MD4403C1_D0026.tif" />

HO ^ = V-NH-PG

О

6b

<img file="MD4403C1_D0027.tif" />

<img file="MD4403C1_D0028.tif" />

V-NH-PG

Scheme 6 shows a general synthesis of an intermediate of the invention R¹-VC (= O) -PR² wherein, for illustrative purposes, P represents pyrrolidine, R¹ is a generic group which is described as either -E or an amino protecting group, and R² represents a generic group which is described as -WPC (= O) -VE, -WPC (= O) -V-NH-PG, -WP-NH-PG or W-NH-PG. Coupling of amine 6a (or 6d, 6h, 6k) with acid 6b or 6e is performed using a peptide coupling agent (eg HATU) to obtain 6c (or 6f, 6g, 6i, 6j, 61, 6m). , respectively.

MD 4403 Cl 2016.09.30

Scheme 7. Representative synthesis of EVC (= O) -R^x

H₂NVC (= O) -PWPC (= O) -NH-PG V

<img file="MD4403C1_D0029.tif" />

7a

H₂NVC (= O) -PWP-PG

7c

H₂NVC (= O) -PW-PG

7e

H₂NVC (= O) -P-PG

<img file="MD4403C1_D0030.tif" />

<img file="MD4403C1_D0031.tif" />

<img file="MD4403C1_D0032.tif" />

NH-VC (= O) -PWPC (= O) -V-NH-PG ° \ ^7b

NH-VC (= O) -PWP-PG

7g

H₂NVC (= O) -O-PG

7i

<img file="MD4403C1_D0033.tif" />

О

У- NH-VC (= O) -PW-PG ° \ ”о

NH-VC (= O) -P-PG ° x ™ о

NH-VC (= O) -O-PG

Scheme 7 shows a general synthesis of an intermediate of the invention EVC (= O) -R^xwherein, for illustrative purposes, E represents methoxycarbonylamino and R¹ is a generic group that is described as either -PWPC (= O) -V-NH-PG, - PWP-PG, -PW-PG PG, -P-PG or -O-PG. By treating 7a (or 7c, 7e, 7g, 7i) with methyl chloroformate under basic conditions (for example, sodium hydroxide), molecule 7b (or 7d, 7f, 7h, 7j) is obtained -

Scheme 9. Representative synthesis of R^x-PR²

<img file="MD4403C1_D0034.tif" />

Saddle

<img file="MD4403C1_D0035.tif" />

<img file="MD4403C1_D0036.tif" />

Saddle

<img file="MD4403C1_D0037.tif" />

st o

<img file="MD4403C1_D0038.tif" />

Scheme 9 shows a general synthesis of an intermediate of the invention R^x-PR² wherein, for illustrative purposes, R¹ represents -C (= 0) -VE or о protecting group and R² represents a substituted benzimidazole. The formation of benzimidazole is accomplished by coupling

MD 4403 Cl 2016.09.30 of acid 9b or 9e with о arylamine 9a, using a peptide coupling agent, such as HATU, to obtain 9c or 9d. By cyclizing the amide in the presence of an acid (such as acetic acid), the 9d or 9g molecule is obtained which confines benzimidazole.

The formation of multiple benzimidazoles is carried out in the same manner, pumping from bis-diamine to obtain the corresponding bis-benzimidazole.

Scheme 20. Representative synthesis of iV-PWPR²

<img file="MD4403C1_D0039.tif" />

Scheme 20 shows a general synthesis of an intermediate of the invention R ^ PWPR²wherein, for illustrative purposes, R¹ and R² represents independent protecting groups and W represents о unit with two aromatic rings constructed through the mediated cyclization of a transition metal. By alkylating phenol 20b with о alkyl bromide, such as 20a, the ether 20c is obtained. By cyclizing the aromatic ring in the presence of a palladium catalyst, compound 20d is obtained. By treating 20d with CuBr2, potocetone 20e is obtained, which provides 20f upon addition of an acid under basic conditions (eg, Et₃N). By reaction 20f with amine or amine salt (for example, ammonium acetate) the 20g molecule is obtained which confines imidazole. Oxidation of 20g, 20i or 201 can be accomplished by heating in the presence of MnO₂ to obtain 20h, 20j or 20m, respectively. Conversion 20g or 20h with a palladium catalyst, such as Pd₂dba₃ and X-Phos, and о boron source such as fl bis (pinacolato) dibor provides boronic ester 20i or 20j.

The boronic ester is coupled with a suitable coupling partner (eg, 20k) using a palladium catalyst, such as Pd (PPh)₃)₄ or PdCl₂(dppf), to obtain

MD 4403 Cl 2016.09.30

201 or 20m. For each transition metal-mediated cross-coupling reaction, the roles of the nucleophile and the electrophile can be reversed to obtain the same coupling product. Other transitional metal-mediated cross-couplings that allow the construction of W, but include partners and alternative coupling reagents, include, but are not limited to, Negishi, Kumada, Stille, and Ullman bonds. For the preparation of two alternative aromatic rings containing groups W, this general scheme can be applied by the appropriate choice of the initial reagents.

Scheme 21. Representative synthesis of iV-PWPR²

<img file="MD4403C1_D0040.tif" />

<img file="MD4403C1_D0041.tif" />

<img file="MD4403C1_D0042.tif" />

О

<img file="MD4403C1_D0043.tif" />

2Wg

Scheme 21 shows a general synthesis of an intermediate of the invention R ^ PWPR²wherein, for illustrative purposes, R¹ and R² represents independent protecting groups and W represents о unit with two aromatic rings constructed by the mediated cyclization of a transition metal. By treating 20d with an activated vinyl reagent (for example, potassium vinyltrifluoroborate) in the presence of a palladium catalyst (eg, palladium acetate and S-Phos), vinyl compound 21a is obtained. The conversion to the appropriate α-halo ketone can be accomplished by bromination with N-bromsuccinimide, followed by oxidation with MnO.₂. The displacement of the α-halo ketone begins with the addition of an acid under basic conditions (for example, Et₃N). Bromide 21d begins with treatment with pyridinium tribromide and is followed by the addition of a second acid in the base conditions to give diester 21e. By reaction 21e with о amine or amine salt (for example, ammonium acetate) the molecule 21f is obtained which confines imidazole. Oxidation 21f can be carried out in the presence of MnO₂ to obtain 21g.

MD 4403 Cl 2016.09.30

Scheme 22. Representative synthesis of EVC (= O) -PWPR

<img file="MD4403C1_D0044.tif" />

<img file="MD4403C1_D0045.tif" />

<img file="MD4403C1_D0046.tif" />

<img file="MD4403C1_D0047.tif" />

<img file="MD4403C1_D0048.tif" />

Scheme 22 shows a general synthesis of an intermediate of the invention EVC (= 0) PWPR m which, for illustrative purposes, R represents о protecting group and W represents о unit with two aromatic rings. The displacement of the α-halo ketones 21b begins with the addition of an acid under basic conditions (eg Et₃N). Bromide 22b begins with pyridinium tribromide treatment and is followed by the addition of a second acid under basic conditions to obtain diester 22c. By reaction 22c with о amine or amine salt (for example, ammonium acetate) the molecule 22d is obtained which confines imidazole. Oxidation 22d can be carried out in the presence of MnO₂ to obtain 22e.

MD 4403 Cl 2016.09.30

Scheme 23. Representative synthesis of RPWPC (= ()) - EV

<img file="MD4403C1_D0049.tif" />

<img file="MD4403C1_D0050.tif" />

<img file="MD4403C1_D0051.tif" />

Scheme 23 shows a generated synthesis of an intermediate of the invention EVC (= 0) PWPR in which, for illustrative purposes, R represents о protecting group and W represents о unit with two aromatic rings. The displacement of the α-halo ketones 21d continues by the addition of an acid, under basic conditions (e.g., Et₃N). By reaction 23a with о amine or amine salt (for example, ammonium acetate) the molecule 23b is obtained which confines imidazole. Oxidation 23b can be carried out in the presence of MnO₂ to obtain 23c.

Scheme 25. Representative synthesis of EVC (= O) -PWPC (= O) -VE о

H₂NVC (= O) -RWPC (= O) -ve

25

<img file="MD4403C1_D0052.tif" />

О

<img file="MD4403C1_D0053.tif" />

NH-VC (= O) -PWPC (= O) -ve

25b

H₂NVC (= O) -PWPC (= O) -V-NH₂

25c

<img file="MD4403C1_D0054.tif" />

О

<img file="MD4403C1_D0055.tif" />

NH-VC (= O) -PWPC (= O) -V-NH

25d

<img file="MD4403C1_D0056.tif" />

Scheme 25 shows a generated synthesis of a molecule of the invention EVC (= O) -PW-PC (= O) -VE in which, for illustrative purposes, E represents ethylcarbonylamino. By treating either 25a or 25c with one or two equivalents of propionyl chloride respectively under basic conditions (eg sodium hydroxide), molecule 25b or 25d is obtained.

MD 4403 Cl 2016.09.30

Scheme 26. Representative synthesis of EVC (= O) -PR and R ^ PR

PG-P_Xthe о

26

<img file="MD4403C1_D0057.tif" />

<img file="MD4403C1_D0058.tif" />

<img file="MD4403C1_D0059.tif" />

26e

<img file="MD4403C1_D0060.tif" />

26f

Scheme 26 shows a general synthesis of a molecule of the invention EVC (= O) -PR and a molecule of the invention R ^ PR m which, for illustrative purposes, R represents haloimidazole. Imidazole 26b is obtained by treating aldehyde 26a with glyoxal in the presence of ammonium hydroxide. Treatment with either N-bromosuccinamide or iodine gives the corresponding haloimidazole 26c and 26d, respectively. Separation from the corresponding bishalogenated compound can be performed by preparative HPLC. The conversion of bishaloimidazole into mono-haloimidazole can also be accomplished by heating in the presence of sodium sulfite. Subsequent functionalization of group P can be performed after removal of the protecting group and coupling with a suitable acid (EVC (= O) -OH).

MD 4403 Cl 2016.09.30

Scheme 27. Representative synthesis of R^x-PWPR²

<img file="MD4403C1_D0061.tif" />

<img file="MD4403C1_D0062.tif" />

<img file="MD4403C1_D0063.tif" />

Scheme 27 shows an alternative alternative general synthesis of an intermediate of the invention R¹PWPR² wherein, for illustrative purposes, R¹ and R² are independent protecting groups and W represents о imitated by two aromatic rings constructed through a mediated cyclization of a transition metal. Bromination 21b with a bromination agent (ie pyridinium tribromide) gives dibromide 27a. The displacement of the primary bromide is then followed by the addition of an acid under basic conditions (for example К₂СОз) to get 21d. Conversion to 21f or 21g can be performed according to the methods described in Scheme 21.

MD 4403 Cl 2016.09.30

<img file="MD4403C1_D0064.tif" />

<img file="MD4403C1_D0065.tif" />

<img file="MD4403C1_D0066.tif" />

<img file="MD4403C1_D0067.tif" />

Scheme 28 shows a general alternative о synthesis of an intermediate of the invention EV-C (= O) -PWPR wherein, for illustrative purposes, R represents о protecting group and 10 W represents о unit with two aromatic rings. Bromination 21b with a bromination agent (ie pyridinium tribromide) gives dibromide 27a. The movement of the primary bromide is then preceded by the addition of an acid under basic conditions (for example К₂СОз) to obtain 22d. The transformation into 22d or 22e can be carried out in accordance with the methods described in Scheme 22.

Specific embodiments

In one embodiment, the invention provides a compound having the formula:

<img file="MD4403C1_D0068.tif" />

or о its pharmaceutically acceptable salt.

MD 4403 Cl 2016.09.30

The invention is further illustrated by the following non-limiting Examples.

The following abbreviations are used in the description of the invention, including in the Examples.

<td>(Aq)</td><td>Aqueous</td>

<td>(G)</td><td>Gaseous</td>

<td>(S)</td><td>Solid</td>

<td>° C</td><td>Celsius degrees</td>

<td>Needle</td><td>Acetate</td>

<td>ACN</td><td>Acetone itril</td>

<td>apprx</td><td>approximately</td>

<td>Bis-pinB / (bp)₂/ (PinB)₂</td><td>Bis (pinacolato) diboron</td>

<td>BOC / Boc</td><td>tert-Butoxycarbonyl</td>

<td>Calc'd</td><td>calculating</td>

<td>CC50</td><td>50% Cytotoxicity concentrates</td>

<td>COMMUNICATION</td><td>1 - [(1 - (Cyano-2-ethoxy-2oxoethylideneaminooxy) dimethylaminomorpholine)] uronium hexafluorophosphate</td>

<td>d</td><td>Doublet</td>

<td>dba</td><td>dibenzalacetonă</td>

<td>DCM</td><td>dichloromethane</td>

<td>dd</td><td>Doubles doubles</td>

<td>ddd</td><td>Doubles doubles doubles</td>

<td>DIPEA / DIEA</td><td>Ν, Ν-Diisopropylethylamine</td>

<td>DMA</td><td>Ν, Ν-dimethylacetamide</td>

<td>DMAP</td><td>4-Dimethylaminopyridine</td>

<td>DME</td><td>dimethoxyethane</td>

<td>DMEM</td><td>Eagle's minimal essential environment</td>

<td>DMF</td><td>Dim ethyl amide form</td>

<td>DMSO / DMSO</td><td>dimethylsulphoxide</td>

<td>dppf</td><td>1, l'-bis (diphenylphosphanyl) ferocene</td>

<td>dt</td><td>Triple doubles</td>

<td>what the₅₀</td><td>Maximum effective semi-concentration</td>

<td>IEP</td><td>lonization through the electroplating ver</td>

<td>et</td><td>Ethyl</td>

<td>ext.</td><td>searing</td>

<td>SFB</td><td>Be a fetal bovine</td>

<td>g</td><td>Gram</td>

<td>HATU</td><td>2- (H-7-Azabenzotriazol-l-yl) -l, l, 3,3-tetramethyl uronium hexafluorophosphate Methanamine</td>

<td>HPLC</td><td>High performance liquid phase chromatography</td>

<td>hr / li</td><td>Hour</td>

<td>Hz</td><td>Herta</td>

<td>J</td><td>Coupling constant (link)</td>

<td>SMCL</td><td>Mass spectrometry of liquid chromatography</td>

<td>M</td><td>Molar</td>

<td>rn</td><td>multiplet</td>

<td>m / z</td><td>Table for loading</td>

<td>M +</td><td>Material point</td>

<td>Me</td><td>Methyl</td>

<td>mg</td><td>Milligram</td>

<td>MHz</td><td>megahertz</td>

<td>min</td><td>Minute</td>

<td>mL</td><td>Milliliter</td>

<td>mmol</td><td>millimoles</td>

<td>boor</td><td>methoxycarbonyl</td>

MD 4403 Cl 2016.09.30

<td>THX</td><td>Mass spectrometry</td>

<td>MTBE</td><td>Methyl tert-butyl ether</td>

<td>N</td><td>Normal</td>

<td>NADPH</td><td>Nicotinamide adenine dinucleotide phosphate</td>

<td>NBS</td><td>I don't brag about the idea</td>

<td>NMM</td><td>N-methylmorpholine</td>

<td>NMR</td><td>Nuclear magnetic resonance</td>

<td>o / n</td><td>Over night</td>

<td>Papp</td><td>Apparent permeability</td>

<td>PBS</td><td>Phosphatic buffer system</td>

<td>Pd / C</td><td>Palladium on carbon</td>

<td>F</td><td>Phenyl</td>

<td>Fg / FGli</td><td>phenylglycine</td>

<td>Piv</td><td>pivalate</td>

<td>Pro</td><td>proline</td>

<td>twitch</td><td>pyridine</td>

<td>cv</td><td>quartet</td>

<td>CVD</td><td>Quartet of doubles</td>

<td>edge</td><td>Quantitative</td>

<td>quint</td><td>quintet</td>

<td>TC / TC</td><td>Room's temperature</td>

<td>s</td><td>singlet</td>

<td>SFos</td><td>2-dicyclohexylphosphino-2 ', 6'-dimethoxy</td>

<td>t</td><td>Triplet</td>

<td>t-Bu</td><td>tert-Butyl</td>

<td>TEMPO</td><td>(2,2,6,6-Tetramethyl-piperidine-1-yl) oxyl</td>

<td>tf</td><td>Tr ifluoromethane sulfon at</td>

<td>ATF</td><td>Trifluoroacetic acid</td>

<td>THF</td><td>tetrahydrofuran</td>

<td>Tr</td><td>threonine</td>

<td>css</td><td>Thin layer chromatography</td>

<td>tol.</td><td>toluene</td>

<td>UV</td><td>Ultraviolet</td>

<td>Oh my</td><td>valine</td>

<td>w / v</td><td>Weight by volume</td>

<td>w / w</td><td>Weight by weight</td>

<td>X-Fos / Xpose / Xfos</td><td>2-Diciclohexilfosfmo-2 ', 4', 6'-triizopropilbifenil</td>

<td>δ</td><td>Chemical displacement</td>

<td>Figure</td><td>Microgram</td>

<td>pL</td><td>microliter</td>

MD 4403 Cl 2016.09.30

EXAMPLES

Example LQ

<img file="MD4403C1_D0069.tif" />

<img file="MD4403C1_D0070.tif" />

Womo-z-tfrctm cm etii&gt;. 4-С.10ГЬ € П2йПа

Ftf (QPw) ^ ż-BuCOșH _r:

K₂CO₃. DMA. 6Ό ° C, also 24

67- 85%

<img file="MD4403C1_D0071.tif" />

9-Ьгото-з-iioro 'GI T-aifrictro-sH ftii&gt; eniojc_:gfefomen-8i.3l4} -one

K2CO3, DMF rt. 18 hours

89%

<img file="MD4403C1_D0072.tif" />

3-chloro-1 ιί.ΙΊτά ^ ιϊάΓΟ-δΗtiiibe пго (с_;д? сг^:0.п) ёп-8 (9H&gt; one

<img file="MD4403C1_D0073.tif" />

⁰ Boe

D1REA, СН₃СЧ 50'У a / NHiChToluen _f

2-JTi etcxItanci! _r1WX

<img file="MD4403C1_D0074.tif" />

? - (2-bromo-5-chlorofcerzyloxyl • 3,4-di; hydrc&gt; nattale-.n-1 (2H} -on a

Cu3p₂r^:CHC ^ £ t0Ac '“б 2 лге

- 95%

<img file="MD4403C1_D0075.tif" />

tert-Bull! 2- (9-010 (0-14,5,11tstfahHrOizQcrQmenoH ^ iei'Tlnaito il; 3-diimr ^^ Ql - 2U:&gt; pîro)^:IÎdin them: -caTb0xiJâtâ

MD 4403 Cl 2016.09.30

<img file="MD4403C1_D0076.tif" />

P ^ DBS₃, dioxane / 90 ° С

<img file="MD4403C1_D0077.tif" />

t: ert-buți '!, 2-f9 ^ lprc- ~ i'hjdroi z осгатето [1: 24φ'ιή ^ 3Ϊ®24ί} ρΐίόΙ idin-Lcarboxiiata

<img file="MD4403C1_D0078.tif" />

(tert ^ Litil ÂД / â.-tetramstif 1.3,2 / iQxabotel^:year - 24L;} -.

1..14 Ш: к} ге12Ьс (эж ^ tpfidin-'i-cîboxij.ață

<img file="MD4403C1_D0079.tif" />

t ^ rt-btitfț 2 - [9-i241 ~ | h .- (rt ^ toxipaHjo-rti ί ^ ΙΐΙ] ρίΓθί ^ ΐίΐ'24 / ϊΗ-. '' 1тШгрН45} -1Л5&gt; 4 | М '' naphtha [1 , .2-d JiraidaibbS-illpifplidiri¹ 1-cafbnxiiată.

HCk EtPH&gt; 6b * C-

<img file="MD4403C1_D0080.tif" />

GGiMU, plPEĂ, DMF, RT

<img file="MD4403C1_D0081.tif" />

acidification! 1 - (2-ξ5-ρ-Π kic ărbonîl} ajnihQ] (feniî} a0et ig ^ pÎFdiidînă ^ ilFl &lt;1'teihidroizpcrQn ^_:eno [4 \: 3 '; S ·,? tn • heads_; 2 *: .dJimid32.0H-ji FlH-imkl-azdΙΊί ^ τηβίίΜ ₇,: oxQbutari -2-ilIca^;Men: small

7- (2-bromo-5-chlorobenzyloxy) -3,4-dihydro-naphthalene-l (2H) -one

To a stirred solution of 7-hydroxy-1-tetralone (13.9 g, 85.7 mmol) and l-bromo-2 (bromomethyl) -4-chlorobenzene (25.6 g, 90.0 mmol) in dimethylformamide ( 850 mL) was added potassium carbonate (24 g, 172 mmol). The reaction product was stirred in an argon atmosphere for 18 hours, then diluted with ethyl acetate (1 L). The organic layers are washed three times with water and о with saline. The organic layer is then dried with magnesium sulfate, filtered and concentrated. Methanol (500 mL) was added to the resulting oil and the suspension was stirred for thirty minutes. 7- (2-bromo-5-chlorobenzyloxy) -3,4-dihydro-naphthalene-1 (2H) -one (27.8 g, yield is 89%) was isolated by filtration.

3-chloro-10, ll-dihydro-5H-dibenzo [c, g] chromo-8 (9H) -one

In a 1 L vial containing palladium pivalate (II) (1.18 g, 3.8 mmol), tri (4fluorophenyl) phosphorus (1.20 g, 3.8 mmol), pivalic acid (2.33 g , 22.8 mmol) and potassium carbonate (31.8 g, 228 mmol) were added a solution of 7- (2-bromo-5-chlorobenzyloxy) -3,4-dihydro-naphthalene-1 (2H) -one (27 , 8 g, 76.2 mmol) in dimethiacetamide (380 mL).

MD 4403 Cl 2016.09.30

The vial is evacuated and refilled with argon 5 times and then stirred in argon atmosphere at 60 ° C for 24 hours. The reaction product is cooled to room temperature and diluted with MTBE and water. The resulting two-phase mixture is stirred for 3 hours and filtered through Celite, then rinsed with MTBE. The organic layer of the filtrate is separated and then washed twice with water and о with brine. The organic layers are then dried with magnesium sulfate, filtered, concentrated and purified by flash column chromatography (Hexanes / DCM) to give 3-chloro-

10.11- dihydro-5H-dibenzo [c, g] chromo-8 (9H) -one (14.4 g, yield is 67%) as a gray-white solid.

9-bromo-3-chloro-10, ll-dihydro-5H-dibenzo [c, g] chromo-8 (9H) -one

To a mixture of 3-chloro-10 1-dihydro-5H-dibenzo [c, g] chromo-8 (9H) -one (14.8 g, 52 mmol) in chloroform (50 mL) and ethyl acetate ( 50 mL) was added copper (II) bromide (24.3 g, 104 mmol). The reaction product is heated to 80 ° C for 2 hours and then cooled to room temperature. The mixture was diluted with dichloromethane and washed twice with 5: 1 solution of aqueous saturated sodium chloride and aqueous ammonium hydroxide (~ 38%), and washed with water. The organic layer was dried with magnesium sulfate, filtered and concentrated to give 9-bromo-3-chloro-10,11-dihydro-5H-dibenzo [c, g] chromo-8 (9H) -one (18.5 g, the yield is&gt; 95%); purity of&gt; 95%.

Note: This reaction is not always clean. Sometimes there is over-bromination, and sometimes there is significant raw material. These impurities can be removed by flash column chromatography.

tert-Butyl 2- (9-chloro-1,4,5-tetrahydroisocromeno [4 ', 3': 6,7] naphtho [1,2d] imidazol-2-yl) pyrrolidine-1-carboxylate

At о solution of (lR) -2- (tert-butoxycarbonyl) cyclopentancarboxylic acid (10.17 g, 47.25 mmol) and 9-bromo-3-chloro-10, l-dihydro-6H-naphtho [2, 3-c] chromo-8 (9H) -one (5.7 mg, 15.7 mmol) in acetonitrile (50 mL) was added diisopropylethylamine (11.11 mL, 64 mmol). The reaction product was stirred at 50 ° C for 4 hours and then diluted with ethyl acetate. The organic layers are washed with water and brine, dried (MgSO₄) and concentrate. The resulting crude residue was purified by flash chromatography to afford (2S) -l-tert-butyl 2- (3-chloro-8-oxo-8,9,10, 1-tetrehhydro-5H-naphtho [c, g] chromen-9-yl) pyrrolidine-1,2-dicarboxylate (4.52 g, 58%). To the solution of (2S) -l-tert-butyl 2- (3-chloro-8-oxo-8,9,10, 1-tetrahydro-6H-naphtho [2,3-c] chromo-9-yl) pyrrolidine To 1, 2-dicarboxylate (3.27 mg, 6.56 mmol) in a mixture of toluene (11 mL) and 2-methoxyethanol (0.7 mL) was added ammonium acetate (5.06 g, 65.6 mmol) ). The reaction mixture is heated to 110 ° C for 3 hours, cooled to room temperature and diluted with ethyl acetate. The organic layers are washed with water and saline solution, dried (Na₂SO₄) and concentrate. The crude residue was purified by flash chromatography to give tert-butyl 2- (9-chloro-1,4,5,1-tetrehhydroisocromeno [4 ', 3': 6,7] naphtho [1,2-d] imidazole-2-yl) pyrrolidine-1-carboxylate (1.95 g, 61%). LCMS-ESI⁺: calculated for C₂₇H₂₈C1N₃O3₄₂: 477.98; observed [M + l]⁺: 478.47 tert-Butyl 2- (9-chloro-1,1-dihydroisocromeno [4 ', 3': 6,7] naphtho [1,2-d] imidazol-2yl) -pyrrolidine-1-carboxylate

The solution of tert-butyl 2- (9-chloro-1,4,5,1-tetrehhydroisocromeno [4 ', 3': 6,7] naphtha [1,2-d] imidazol-2-yl) - pyrrolidine-1-carboxylate (1.9 g, 3.96 mmol) in dichloromethane (35 mL) was added manganese (IV) oxide (17 g, 198 mmol). The reaction mixture was stirred at room temperature for 18 hours, then diluted with ethyl acetate. The organic layers are washed with water and saline solution, dried (Na₂SO₄) and concentrate. The crude residue was purified by flash chromatography to give tert-butyl 2- (9-chloro-

1,11-dihydroisocromeno [4 ', 3': 6,7] naphtho [1,2-d] imidazol-2-yl) pyrrolidine-1-carboxylate (1.52 g, 81%). LCMS-ESU: calculated for C₂₇H₂₆C1N₃O3₄₂: 475.9; observed [M + l]⁺: 476.45.

tert-Butyl 2- [9- (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) -l, lhydroisocromeno [4 ', 3': 6,7] naphtha [1, 2-d] imidazol-2-yl] pyrrolidine-l-carboxylate

A degassed mixture of tert-butyl 2- (9-chloro-1, lldhydroisocromeno [4 ', 3': 6,7] naphtho [1,2-d] imidazol-2-yl) -pyrrolidine-1-carboxylate (1 , 52 g, 3.17 mmol), bis (pinacolato) boron (1.21 g, 4.75 mmol), potassium acetate (934 mg, 9.52 mmol), tris (dibenzylideneacetone) palladium (116 mg, 0 , 13 mmol) and 2-dicyclohexylphosphine 2 ', 4', 6'-tri-i-propyl-1, r-biphenyl (121 mg, 0.08 mmol) in 1,4-dioxane (16 ml) is heated

MD 4403 Cl 2016.09.30 to 90 ° C for 1.5 hours, cool to room temperature and dilute with ethyl acetate. The organic layers are washed with water and brine, dried (Na₂SO₄) and concentrate. The crude residue was purified by flash chromatography to give tert-butyl 2- [9- (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) -1,1-dihydroisocromeno [4 ' , 3 ': 6,7] naphtho [1,2-d] imidazol-2-yl] pyrrolidine-1-carboxylate (1.7 g, 94%) tert-Butyl 2- [9- (2- {1 [N- (methoxycarbonyl) valyl] pyrrolidine-2-yl} -LH-imidazol-5-yl) LJL-dihidroizocromeno [4 ', 3': 6,7] naphtho [l, 2-d] imidazol-2-yl ] pyrrolidine-lcarboxylatul

To a solution of methyl (S) -l - ((S) -2- (5-bromo-1H-imidazol-2-yl) pyrrolidine-1-yl) -3-methyl-1-oxobutan-2-ylcarbamate (1, 48 g, 3.97 mmol), tert-butyl 2- [9- (4,4,5,5-tetramethyl, 3,2-dioxaborolan-2-yl) -l, II-dihydroisocromenon [4 ', 3' : 6,7] naphtho [1,2-d] imidazol-2yl] pyrrolidine-1-carboxylate (1.88 g, 1.48 mmol), tetrakis (triphenylphosphine) palladium (0) (191 mg, 0.16 mmol ) and dichloro [l, r-bis (diphenylphosphine) ferrocene] palladium (II) (242 mg, 0.33 mmol) in a mixture of 1,2-dimethoxyethane (37.0 mL) and dimethylformamide (6 mL) was added to a potassium carbonate solution (2M in water, 5 mL, 9.93 mmol). The resulting mixture is degassed and then heated to a temperature of 85 ° C in an argon atmosphere for 18 hours. After cooling to room temperature, the reaction mixture was diluted with ethyl acetate. The organic layers are washed with water and brine, dried (Na₂SO₄) and concentrate. The crude residue was purified by flash chromatography to give tert-butyl 2- [9- (2- {1- [N- (methoxycarbonyl) valyl] pyrrolidine-2-yl} -Himidazol-5-yl) -1,11 -dihydroisocromeno [4 ', 3': 6,7] naphtho [1,2-d] imidazol-2-yl] pyrrolidine-1 carboxylate (1.45 mg, 59%). LCMS-ESI⁺: calculated for QiH ^ C ^ 733.86; observed [M + l]⁺: 734.87.

[1- (2- {5- [2- (1 - {[(Methoxycarbonyl) amino] (phenyl) acetyl} pyrrolidine-2-yl) -l, lhydroisocromeno [4 ', 3': 6,7] naphthoic acid [1, 2-d] imidazol-9-yl] -H-imidazol-2-yl} pyrrolidine-1-yl) -3-methyl-1-oxobutan-2-yl] carbamic

О tert-butyl solution 2- [9- (2- {1- [N- (methoxycarbonyl) valyl] pyrrolidine-2-yl} -Himidazol-5-yl) -1,11-dihydroisocromeno [4 ', 3' : 6,7] naphtho [1,2-d] imidazol-2-yl] pyrrolidine-1 carboxylate (462 mg, 0.63 mmol), ethanol (6 mL) and concentrated hydrochloric acid (2 mL) are heated to 60 ° C for 1 hour. The reaction product is concentrated and the crude material is dissolved in DCM (6 mL). The solution is concentrated and a solution of (R) -2- (methoxycarbonylamino) -2-phenylacetic acid (172 mg, 0.82 mmol) and COMU (311 mg, 073 mmol) in DMF (6 mL) are added to this material. ). Diisopropylethylamine (330 µL, 1.89 mmol) is added to the resulting solution. After stirring for 18 hours at room temperature, the reaction mixture was diluted with ethyl acetate, washed with water and brine, dried (Na₂SO₄), concentrated and purified by reversible phase HPLC (Gemini, between 15 and 45% ACN / H₂O + 0.1% TFA). The product fractions are lyophilized to obtain [1- (2- {5- [2- (1 {[(methoxycarbonyl) amino] (phenyl) acetyl} pyrrolidine-2-yl) -l, lhydroisocromeno [4 ', 3') : 6,7] naphtho [1,2-d] imidazol-9-yl] -lH-imidazol-2-yl} pyrrolidine-yl) -3-methyl-1-oxobutan-2-yl] carbamic (231 mg, 45%). LCMS-ESI⁺: calculated for CAN ^: 824.92; observed [M + l]⁺: 826.00

MD 4403 Cl 2016.09.30

Example OF

<img file="MD4403C1_D0082.tif" />

depotassium /.

Pd (OAc) 2, SPhos, K7CO3

1. KBS к₂а.тн? / ам $ о proparwi (reilux)

<img file="MD4403C1_D0083.tif" />

3-'Зп 1G. 11 -d id id ro-SH -.

: 0ίΐ&gt; 6υΣ.^:θΙθ;_;3,: ύΐΌΒ ·· &amp; η-3ΐ9Η? - on ă.

<img file="MD4403C1_D0084.tif" />

<img file="MD4403C1_D0085.tif" />

Cs_?CO_s

2-Me-WF lA-brc моасеЖ1Д 11 id rc-5H-.

ci id * n 2 c {c, g} его mș - δ fȘ-Hon â iroiidin-2 'carboxylic

<img file="MD4403C1_D0086.tif" />

q: r; d.iniumir: op ^): d ·

DCMfMeOH (2Ș; 4SM-ted-buti! 2- (2-gxo ^ 2-i'8-o.xo -8.9.1 ΰ_:11_TtetraH idr0-SH -d ·!&gt; ΐ? ηzq-fς..π -.3 - iltetii} 4- (r.frtoxirrjistiÂiroiidin-i ₍2-d! Ca &lt;box!: 8: t

<img file="MD4403C1_D0087.tif" />

; 2S.4S 3-2-Ț2- (9-bro mo - &amp; - oxo-AA1 δ.? - i₇1е1гэЙ'Йго-5Йd: ti: NZC c_fg;: c.rorr ^ n-3 -: {N2 - (? xGitii '1 -fert-b useful imeioxin ^ niip.ifoliOin ^ l .S-dicarboxitai

<img file="MD4403C1_D0088.tif" />

; ackl · f2S.S $ ^ 1- (f £ &lt;-2 '(met о klc 8 rd о n-ia гг «&gt; п о&gt; -3meiM ano 3} -S- ·? &amp; £!? ire:' β ϊ n s-2 carboxylic acid

<img file="MD4403C1_D0089.tif" />

w &amp; tjibctflfioijyE- ^ ipkolidin- ^ ca.teb- ^ ic-xiV ^ c-xG ^. &amp; JX ^ I 1-tetrahydro-.SHd: ibenz.o. [c, g &lt;crc.rți6n-3-: i:_;ş-2-oxO € t4; · 4- (metGxi: ^ «: pif5 îioin-1-, 2dicarb oxter

MD 4403 Cl 2016.09.30

<img file="MD4403C1_D0090.tif" />

TA-rt-biîT :! : (2S: 4S) -2- · S ~ · 2A 2S, 5S} -14ti-i \ n&gt; eto-x^:: c8Fbc-ailj-L-AA-. .. teirflLhtf roizp was 3 A. 'Tjjha йо {1_:2Ai iHnaî d:? - SA; $ -.

1Η -: ΓΠί03ίόΓ2-: ζ: -4- !; AcioxiîTistîi? Pîroikiin-1 -ca rscx;&gt; aî

<img file="MD4403C1_D0091.tif" />

d ih id-r &amp; izt cr0m ^ nc: 4 \ 3^?i^:. Ti ^ aftc · :! , 2A} ihiiq^:3.Ξ.όϊ-&gt; H-IFN Μ ·}: dazGi-2-yl] A-i'rn &amp; toxir: -: ethyl!) C! Re-oxopyrrolidin - 1-ii3rbGxiia i

1, HCiMioxân '; DCM

2. HAW., DIPEA, DMF

О _ΪΛΧ. JbIHCOsMe

НО * У * ····. . .

/ -3CEÎ (2S.2S-A-2Ч йТ: 01ах1ра: гЬ0пватюо^; • Ș-mHȘpienianolC '

<img file="MD4403C1_D0092.tif" />

пзе11А (2 $_:3 £ / 1; А2 ^ ftmrtaitartiG-n il} amincE / fi^v"tilbuta.n _;Г1 ~.

ih ksrsizo сгс'.тйпоА ··, 3;&gt; S, 7} na fte-ί Г. 2 iiH-f ^ tDXiGTsi ^ iDirariin-I ^^ S-iTifeiir'L &amp; xbp &amp; ntan-z-iOcarbarriai

3-vinyl-10, ll-dihydro-5H-dibenzo [c, g] chromen-8 (9H) -one

A 500 ml round-bottomed, triple-necked dry flask in an oven is cooled with Ar, then charged with 3-chloro-10, 1-dihydro-5H-dibenzo [c, g] chromo-8 (9H) -one (12.0 g, 42.1 mmol), potassium vinyltrifluoroborate (8.47 g, 6.32 mmol), Pd (OAc)₂ (473 mg, 2.11 mmol), SPhos (1.74 g, 4.25 mmol), K₂CO₃ (17.5 g, 126 mmol) and anhydrous propanol (120 mL). The reaction mixture was bubbled with Ar for 16 minutes, then heated at reflux for 5.5 hours. After opening, the reaction mixture is cooled to room temperature and concentrated under reduced pressure. The crude residue is suspended in DCM, then washed with H₂A saline solution. The organic solution is dried over MgSO₄, filter and concentrate under reduced pressure. The resulting residue was further purified by silica buffer, eluting with DCM to afford 3-vinyl-10.11-dihydro-5H-dibenzo [c, g] chromo-8 (9H) -one (10.2 g, 87%).

3- (2-bromoacetyl) -10, ll-dihydro-5H-dibenzo [c, g] chromen-8 (9H) -one

3-Vinyl-10, II-dihydro-5H-dibenzo [c, g] chromo-8 (9H) -one (9.98 g, 36.1 mmol) was dissolved in a stirred solution of THF (70 mL) , DMSO (70 mL) and H₂O (35 mL). NBS (6.75 g, 37.9 mmol) was added in a single porphyry and the reaction mixture was stirred at room temperature for 33 min. At startup, the reaction medium is diluted with EtOAc and washed twice with H₂O and о given with saline solution. The organic phase is dried over MgSO₄, filter and concentrate under reduced pressure. The resulting crude bromohydrin is suspended in DCM (200 mL) and treated with MnO₂ activated (62.7 g, 722 mmol). After stirring for 15 hours at room temperature, the reaction mixture was filtered through celite, and the residue was washed several times with DCM. The combined filtrate (-400 mL) was treated with MeOH (-100 mL) and the mixture was gradually concentrated under reduced pressure, causing the solid material to precipitate from the solution. When the volume of the liquid reaches -200 mL, the solid is filtered and rinsed with MeOH. The sequence of concentration / precipitation / filtration / rinsing is performed 2 more times, resulting in

MD 4403 Cl 2016.09.30 collecting 3 cultures of 3- (2-bromoacetyl) -10, l-dihydro-5Hdibenzo [c, g] chromo-8 (9H) -one (7.49 g, 56%) powder over 2 stages).

(4S) -l- / c / 7-Butyl 2- (2-oxo-2- (8-oxo-8,9,10, ll-tetrahydro-5H-dibenzo [c, g] cromen-

3-I) ethyl) 4- (methoxymethyl) pyrrolidine-1,2-dicarboxylate

3- (2-Bromoacetyl) -10, 1-dihydro-5H-dibenzo [c, g] chromen-8 (9H) -one (7.47 g, 20.1 mmol) and acid (2S, 4S) - (tert -butoxycarbonyl) -4- (methoxymethyl) pyrrolidine-2-carboxylic acid (5.22 g, 20.1 mmol) was suspended m 2-Me-THF (75 mL) and treated with CS2CO3 (3.27 g, 10.1 mmol). After stirring for 4 hours at room temperature, the reaction mixture was diluted with DCM. The organic layer is washed with H₂O. The aqueous layer is re-extracted 2 times with DCM. The combined organic layers are dried over MgSO₄, filter and concentrate under reduced pressure. The crude residue was purified by silica column chromatography (from 10% to 50% EtOAc / DCM) to give (4S) -l-tert-butyl-2- (2-oxo-2- (8oxo-8, 9.10, 1-tetrahydro-5H-dibenzo [c, g] chromen-3-yl) ethyl) -4- (methoxymethyl) pyrrolidine-1,2-dicarboxylate (7.73 g, 70%).

(25.45) -2- (2- (9-bromo-8-oxo-8,9,10, ll-tetrahydro-5H-dibenzo [c, g] cromen-3-yl) -

2-oxoethyl) -l-tert-butyl-4- (methoxymethyl) pyrrolidine-1,2-dicarboxylate (4S) -1-Zer / -Butyl 2- (2-oxo-2- (8-oxo-8.9 , 10, 1,1-tetrahydro-5H-dibenzo [c, g] chromo-3-yl) ethyl) -4- (methoxymethyl) pyrrolidine-1,2-dicarboxylate (7.66 g, 13.9 mmol) was dissolved in a solution of DCM (100 mL) and MeOH (40 mL), then treated with pyridinium tribromide (4.90 g, 15.3 mmol). After stirring at room temperature for 1.75 hours, the reaction mixture was diluted with DCM and washed successively with 10% HC1, saturated aqueous NaHCO solution₃ and saline. The organic phase is dried over MgSO₄, is filtered and concentrated under reduced pressure, and the crude material is still used without further purification.

(2R, 4R) -l- / cr / -Butyl 2- (2- (9 - ((2S, 5S) -l - ((S) -2- (methoxycarbonylamino) -3-methylbutanoyl) -5-methylpyrrolidine-2- carbonyloxy) -8-oxo-8,9,10, ll-tetrahydro-5Hdibenzo [c, g] chromen-3-yl) -2-oxoethyl) -4- (methoxymethyl) pyrrolidine-1,2-dicarboxylate (25.45) -2- (2- (9-bromo-8-oxo-8,9,10, ll-tetrahydro-5H-dibenzo [c, g] chromen-3-yl) -2-oxoethyl) -l-tert-butyl-4 - (methoxymethyl) pyrrolidine-1,2-dicarboxylate (8.76 g, 13.94 mmol) is treated with о solution of (2S, 5S) -I - ((S) -2- (methoxycarbonylamino) -3-methylbutanooyl) 5-methylpyrrolidine-2-carboxylic acid (6.85 g, 23, 92 mmol) in 2-Me-THF (70 mL) and Cs₂CO₃(3.63 g, 11.15 mmol). The stirred reaction mixture was heated to 50 ° C for 20 hours, then cooled to room temperature and diluted with EtOAc. The organic phase is washed with H₂An saline solution and then dried over MgSO₄, filter and concentrate under reduced pressure. The crude residue is purified by silica column chromatography (from 0% to 30% MeOH / AcOEt) to give (2R, R) -IZerr-butyl-2- (2- (9 - ((2S, 5S ) -l - ((S) -2- (methoxycarbonylamino) -3-methylbutanyl) -5-methylpyrrolidine-2-carbonyloxy) -8-oxo-8,9,10,1-tetrahydro-5H-dibenzo [c, g] chromen-3-yl) 2-oxoethyl) -4- (methoxymethyl) pyrrolidine-1,2-dicarboxylate (10.47 g, 90%).

ZerZ-Butyl (2S, 4S) -2- [5- (2 - {(2S, 5S) -l- [N- (methoxycarbonyl) -L-valyl] -5-methylpyrrolidine-2-yl} -l, 4,5 , II-tetrahydroisocromeno [4 ', 3': 6,7] naphtho [1,2-d] imidazol9-yl) -1H-imidazol-2-yl] -4- (methoxymethyl) pyrrolidine-1-carboxylate (2R, 4R) -1-Zerr-Butyl 2- (2- (9 - ((2S, 5S) -l - ((S) -2- (methoxycarbonylamino) -3-methylbutanoyl) -5-methylpyrrolidine-2-carbonyloxy) -8- oxo-8,9,10,1-tetrahydro-5H-dibenzo [c, g] chromen-3-yl) -2-oxoethyl) -4- (methoxymethyl) pyrrolidine-1,2-dicarboxylate (10.47 g, 12 , 56 mmol) and NH₄OAc (50.9 g, 660 mmol) was suspended in a solution of 10: 1 PhMe / 2-methoxyethanol (132 mL). The stirred reaction mixture was heated to 110 ° C for 4.5 hours, then cooled to room temperature and diluted with EtOAc. The organic phase is washed 3 times with saturated aqueous NaHCO₃, then dried over MgSO₄, filter and concentrate under reduced pressure. The crude residue is purified by silica column chromatography (from 0% to 30% MeOH / EtOAc) to give-butyl (2S, 4S) -2- [5- (2 - {(2S, 5S) - L- [N- (methoxycarbonyl) -L-valyl] -5metilpirolidină-2-yl} -l, 4,5, ll-tetrahidroizocromeno [4 ', 3': 6,7] naphtho [l, 2-d] imidazole -9-yl) 1H-imidazol-2-yl] -4- (methoxymethyl) pyrrolidine-1-carboxylate (8.33 g, 84%).

tert-Butyl (2S, 4S) -2- [5- (2 - {(2S, 5S) -l- [N- (methoxycarbonyl) -L-valyl] -5-methylpyrrolidine-2-yl} -l, II-dihydroisocromenon [4 ', 3': 6,7] naphtho [l, 2-d] imidazole-9-yl) H-imidazol-2-yl] -4- (methoxymethyl) pyrrolidine-l-carboxylate

MD 4403 Cl 2016.09.30 terZ-Butyl (2S, 4S) -2- [5- (2 - {(2S, 5S) -l- [N- (methoxycarbonyl) -L-valyl] -5-methylpyrrolidine-2-yl) -1,4,5,1-tetrahydroisocromeno [4 ', 3': 6,7] naphtho [1,2-d] imidazol-9-yl) 1H-imidazol-2-yl] -4- (methoxymethyl) pyrrolidine-1-carboxylate (8.33 g, 1.049 mmol) is suspended in DCM and added in a single MnO portion₂ activated (55.0 g, 630 mmol). After 13 hours MeOH (200 mL) was added and the suspension filtered through celite. The residue was washed with MeOH (600 mL) and the filtrate was concentrated under reduced pressure. The crude material is purified by silica column chromatography (from 0% to 45% MeOH / EtOAc) to give zerz-butyl (2S, 4S) -2- [5- (2 - {(2S, 5S) - 1- [N (methoxycarbonyl) -L-valyl] -5-methylpyrrolidine-2-yl) -1,11-dihydroisocromeno [4 ', 3': 6,7] naphtho [1,2-d] imidazol-9-yl) -HH-imidazol-2-yl] -4 (methoxymethyl) pyrrolidine-1-carboxylate (4.85 g, 58%).

Methyl {(2S, 3S) -l - [(2S, 4S) -2- (5- {2 - [(2S, 5S) -l - {(2S) -2 - [(methoxycarbonyl) amino] -3-methylbutanyl} -5-methylpyrrolidine-2-yl] -l, ll-dihidroizocromeno [4 ', 3': 6,7] naphtho [l, 2 d] imidazole-9-yl} -LH-imidazol-2-yl) -4- (methoxymethyl) pyrrolidine-1-yl] -3-methyl-loxopentan-2-yl} zinc-butyl carbamate (2S, 4S) -2- [5- (2 - {(2S, 5S) -l- [N- (methoxycarbonyl) -L-valyl] -5-methylpyrrolidine-2-yl) -1,11-dihydroisocromeno [4 ', 3': 6,7] naphtho [1,2-d] imidazol-9-yl) -1Himidazole-2 -yl] -4- (methoxymethyl) pyrrolidine-1-carboxylate (179 mg, 0.226 mmol) was dissolved in DCM (4 mL) and HC1 (4.0 M in dioxane, 1 mL) was added. The reaction mixture was stirred for 1 hour at room temperature, then concentrated under reduced pressure. The resulting residue was treated with (2S, 3S) -2- (methoxycarbonylamino) -3-methylpentanoic acid (51 mg, 0.27 mmol), HATU (95 mg, 0.25 mmol), DMF (2 mL) and DIPEA ( 0.39 mL, 2.3 mmol). After stirring for 6 min, the reaction is quenched with H₂O, filter and purify by reverse phase HPLC to obtain methyl {(2S, 3S) -l - [(2S, 4S) -2- (5- {2 - [(2S, 5S) 1 - {( 2S) -2- [(methoxycarbonyl) amino] -3-methylbutanooyl} -5-methylpyrrolidine-2-yl] -1,11-dihydroisocromeno [4 ', 3': 6,7] naphtha [1,2-d] imidazole- 9-yl} -1H-imidazol-2-yl) -4 (methoxymethyl) pyrrolidine-1-yl] -3-methyl-1-oxopentan-2-yl) carbamate (116 mg, 59%). MS (ESI) m / z 864 [M + H]⁺. Ή NMR (400 MHz, cd₃od) δ 8.57 (d, J = 14.7 Hz, 1H), 8.45 (s, 1H), 8.20 (d, J = 14.4 Hz, 1H), 8.15 - 7, 98 (m, 2H), 7.91 (dd, J = 21.8, 14.1 Hz, 2H), 7.85 - 7.69 (m, 2H), 7.69 - 7.48 (m, 2H), 5.42 - 5.12 (m, 5H), 4.34 (dd, J = 22.3, 13.7 Hz, 1H), 4.30 - 4.10 (m, 2H), 3 , 87 - 3.73 (m, 1H), 3.73 - 3.63 (m, 7H), 3.62 - 3.48 (m, 2H), 3.48 - 3.38 (m, 4H) , 3.35 (s, 3H), 2.95 - 2.70 (m, 1H), 2.70 - 2.55 (m, 2H), 2.55 - 2.20 (m, 2H), 2 , 20-1.91 (m, 3H), 1.77 (d, J = 42.0 Hz, 1H), 1.65 (d, J = 6.6 Hz, 3H), 1.43 (t, J = 24.6 Hz, 1H), 1.28 (d, J = 6.2 Hz, 1H), 1.23 - 1.01 (m, 3H), 0.98 (d, J = 6.6) Hz, 3H), 0.90 (dd, J = 13.1, 5.9 Hz, 10H).

MD 4403 Cl 2016.09.30

Example PY

<img file="MD4403C1_D0093.tif" />

tert-butHH (25,45) -2- (5424 (25,55) -1 oxycarbonylH-vahlFS * metUpiroHdin-2-NНД i-dihydroisocromene {4'3^,R6_f7] n3ftoi1,2 'dJifnidazoI- ^ itblH-imidazQl-Z- ^ M-imetoximetiJlpirolidm1 -rarboxite

1. HC1

2. С0Щ СИРЕА DMF

<img file="MD4403C1_D0094.tif" />

(R) '2. (methoxycarbonitemine) 2-phaoylacelic

<img file="MD4403C1_D0095.tif" />

methyl {{25) -1 - {(25_r55) -2- (942 - {(2S, 4S&gt; 1 - {{2R) -2 [(methoxy-bond) amphna] -2-phenyl-1H (} - 44 OetDxymethyl) pyrrolidin2-yl] -t Hi mHazazone ' 54 H, 11 -di hyd roizocr omeni 44% ': 6,7] naphtha [1,2с5 | т1 &lt;1аго8-2- | 1&gt; 5'те1йриойсйп &lt;1- ||] -' 3-теЬМ-о-хоЬшзп: '2 R} c3rbamat

Methyl {(2S) -l - [(2S, 5S) -2- (9- {2 - [(2S, 4S) -l - {(2R) -2 - [(methoxycarbonyl) amino] -2-phenylacetyl} -4 - (methoxymethyl) pyrrolidine-2-yl] -LH-imidazol-5-yl} -l, lldihidroizocromeno [4 ', 3': 6,7] naphtho [l, 2-d] imidazol-2-yl) -5 methylpyrrolidin-l-yl] -3-methyl-l-oxobutan-2-yl} carbamate

О tert-butyl solution (2S, 4S) -2- [5- (2 - {(2S, 5S) -l- [N- (methoxycarbonyl) -L-valyl] -5-methylpyrrolidine-2-yl) -1, 11-dihydroisocromeno [4 ', 3': 6,7] naphtho [1,2-d] imidazol-9-yl) -1Himidazol-2-yl] -4- (methoxymethyl) pyrrolidine-1-carboxylate (150 mg, 0.19 mmol) in 1.25 N HC1 in EtOH (3 mL) was stirred overnight, then warmed to 50 ° C for 3 hours. The reaction mixture was concentrated and the crude material was dissolved in DMF (2 mL). To this solution is added a solution of (R) -2 (methoxycarbonylamino) -2-phenylacetic acid (52 mg, 0.25 mmol) and COMU (90 mg, 0.21 mmol). Diisopropylethylamine (0.099 mL, 0.57 mmol) is added to the resulting solution. After stirring for 2 hours at room temperature, the reaction mixture was quenched with 1N HCl (0.200 mL) and purified by HPLC. After lyophilization, the TFA salt was dissolved in ethyl acetate and washed with saturated NaHCOH solution. The organic phase is dried over Na₂SO₄ and focus. The free base is then dissolved in MeCN / H₂O and lyophilized to obtain methyl {(2S) -l - [(2S, 5S) -2- (9- {2 - [(2S, 4S) -l - {(2R) -2 [(methoxycarbonyl) amino ] -2-phenylacetyl} -4- (methoxymethyl) pyrrolidine-2-yl] -IH-imidazol-5-yl-1,1-dihydroisocromeno [4 ', 3': 6,7] naphtha [1,2-d] imidazol-2-yl) -5-methylpyrrolidine-l-yl] -

3-methyl-1-oxobutan-2-yl) carbamate (65 mg, 39%). LCMS-ESI⁺: calculated for C49H54N8O8: 882.4; [M + l]⁺ registered: 884.1. Diagnostic points in H-NMR (CD3OD): 8.28 (s, 1H), 8.21 (s, 1H), 8.04 (s, 1H), 7.91 - 7.01 (m, 10H) ), 3.62 (s, 3H), 3.34 (s, 3H), 3.23 (s, 3H), 1.56 (d, 3H), 1.03 (d, 3H), 0.94 (d, 3H).

MD 4403 Cl 2016.09.30

Example PY-1

<img file="MD4403C1_D0096.tif" />

3- {2-bromo-1hydroxyienS) -W, 1 V dehidfo-SHd and benzo Jc.g] cro men ^ ЭН) -опа

<img file="MD4403C1_D0097.tif" />

9-bromo-3q2-bromc-V hydroxyethyl) -1O, 11 'dihydro-5H • dfbenzofcg] cream: 8 (9H&gt; -one

<img file="MD4403C1_D0098.tif" />

9-bramo-342-brom0acetil) '

10_r1 SHdiben2o dihydro-l ^ [£_pGJC: romen8 {9H) -one

9-bromo-3- (2-bromoacetyl) -10, ll-dihydro-5H-dibenzo [c, g] chromen-8 (9H) -one

To 3- (2-bromo-1-hydroxyethyl) -10,1-dihydro-5H-dibenzo [c, g] chromo-8 (9H) -one (20.3 g, 54.4 mmol) in DCM (365 MeOH (22 mL) and pyridinium tribromide (18.24 g, 57.0 mmol) were added. After 2 hours, water (100 mL) is added and after a short stirring the layers are separated and the lower organic layer is collected. The organic layer was then washed with IM HC1 (100 mL) and the lower organic layer containing 9-bromo-3- (2-bromo-1-hydroxyethyl) -10, II-dihydro-5H-dibenzo [c, g] chromene was collected. 8 (9H) -one. 400 MHz Ή NMR (CDC1₃) 7.75 (d, J = 8.1 Hz, 1H), 7.68 (s, 1H), 7.61 (s, 1H), 7.42 (d, J = 7.5 Hz, 1H) , 7.24 (s, 1H), 5.13 (s, 2H), 4.99 - 4.96 (m, 1H), 4.73 (dd, J = 4.1, 4.1 Hz, 1H) ), 3.69 - 3.66 (m, 1H), 3.58 - 3.53 (m, 1H), 3.35 - 3.27 (m, 1H), 2.96 - 2.90 (m , 1H), 2.58 2.44 (m, 2H), C-OH was not observed.

9-bromo-3- (2-bromo-1-hydroxyethyl) -10.1-dihydro-5H-dibenzo [c, g] chromen8 (9H) -one (approx. 54.4 mmol) in DCM (365 Sodium bicarbonate (5.45 g), sodium bromide (6.14 g), TEMPO (16.55 mg) and water (60 mL) are added. The solution was cooled to 0-5 ° C and a bleach of 6% (91.5 mL) was added. After 1 hour isopropyl alcohol (20 mL) is added and the reaction mixture is warmed to room temperature. Stirring is stopped, the layers are separated, then the lower organic layer is collected and concentrated, removing about 345 g of solvent. The slurry was filtered and the residue was washed with 50 mL of water and then with 50 mL of DCM (pre-cooled to 5 ° C). The solid products are collected and dried in vacuo to give 9-bromo-3- (2-bromacetyl) -10, II-dihydro-5H-dibenzo [c, g] chromen-8 (9H) -one (18.6 g , with a yield of 76%). 400 MHz^TH NMR (CDC1)₃) δ 8.03 - 8.01 (m, 1H), 7.85 (d, J = 8.2 Hz, 1H), 7.82 (s, 1H), 7.71 (s, 1H), 7 , 67 (s, 1H), 5.19 (s, 2H), 4.74 (dd, J = 4.1, 4.1 Hz, 1H), 4.45 (s, 2H), 3.37 - 3.29 (m, 1H), 2.99 - 2.92 (m, 1H), 2.59 - 2.46 (m, 2H); 100 MHz 13C NMR (CDC1₃) δ 190.4, 189.6, 154.2, 136.6, 134.1, 133.9, 132.9, 131.8, 129.3, 127.2, 125.6, 124.2, 123.3, 117.0, 68.1, 49.9, 31.8, 30.4, 25.5.

Example PY-2

<img file="MD4403C1_D0099.tif" />

<img file="MD4403C1_D0100.tif" />

3- (2-b ro moacetyl&gt; - 10,11dihydro-5Hdi be nzo [c, g] c ro m en ~

9-bromo-3- (2-bromoacetyl) -W, 11dihydro ^ hl · of benzofcgjcro m en3 {9H) -one

9-bromo-3- (2-bromoacetyl) -10, ll-dihydro-5H-dibenzo [c, g] chromen-8 (9H) -one

A mixture of 3- (2-bromacetyl) -10, 1-dihydro-5H-dibenzo [c, g] chromo-8 (9H) -one (2.58 g, 6.95 mmol), pyridinium tribromide ( 2.56 g, 8.0 mmol), dichloromethane (22 mL) and methanol (2.5 mL) were stirred at about 20 ° C for 3 hours to obtain a precipitate. The precipitated product was filtered, washed with dichloromethane (10 mL) and dried in a vacuum oven at 40 ° C to obtain 9-bromo-3- (2-bromacetyl) -10, 1-dihydro-5H- dibenzo [c, g] chromen-8 (9H) -one (2.62 g, in a yield)

MD 4403 Cl 2016.09.30 of 84%). 400 MHz NMR (CDC1₃) δ 8.03 - 8.01 (m, 1H), 7.85 (d, J = 8.2 Hz, 1H), 7.82 (s, 1H), 7.71 (s, 1H), 7 , 67 (s, 1H), 5.19 (s, 2H), 4.74 (dd, J = 4.1, 4.1 Hz, 1H), 4.45 (s, 2H), 3.37 - 3.29 (m, 1H), 2.99 - 2.92 (m, 1H), 2.59 - 2.46 (m, 2H).

Example PY-3

Cl

<img file="MD4403C1_D0101.tif" />

<img file="MD4403C1_D0102.tif" />

65*0

3-doro4 0J1 -dihforo

5HP .................

) PdCyMeCNfe'X'Phos ^{T 3} ™ K3PO4, MeCN, 65 C 34tnmet8lsi¨) etim1&gt;

W, 11-dihydro-5Hdi benzole, g 8 g (9H) -one

<img file="MD4403C1_D0103.tif" />

3-acet8l-1O, 11-dihydrrQ5Hd ibenzQ [c, g] c ro me n8 (9H) -one d: ibenzoic, g] € FomenPyHBr ^

DCM? MeOH ^cC

8 (9H) -one

<img file="MD4403C1_D0104.tif" />

9-bromo-3- (2-bromoacetib-10,11 dihydro-SH ^ ibenzo KgcromenB (9H) -one

3 - ((trimethylsilyl) ethynyl) -10, ll-dihydro-5H-dibenzo [c, g] chromen-8 (9H) -one

A 3 00 mL flask fitted with a suspended magnetic stirrer and a reflux condenser in a nitrogen atmosphere is charged with 3-chloro-10,11-dihydro-5Hdibenzo [c, g] chromo-8 (9H) -one (10.0 g, 35.12 mmol), anhydrous triphasic phosphate powder (22.4 g, 105.4 mmol), XPhos (1.34 g, 2.81 mmol) and PdCl₂(MeCN)₂ (364 mg, 1.40 mmol). Acetonitrile (140 mL) was added, followed by TMSacetylene (18 mL, 141 mmol). The mixture is heated to 65 ° C. After 6 hours, the reaction is considered complete and the mixture is cooled to 20 ° C. The mixture is filtered through a fried funnel, and the precipitate on the filter is washed with acetonitrile. The filtrate is concentrated to about 150 mL under reduced pressure and extracted with heptane (50 mL, 3x100 mL). N-acetylcysteine ​​(15 g) was added to the acetonitrile phase and the mixture was stirred for 5 hours at 45 ° C. The mixture is cooled to Ipana at room temperature, filtered through a fried funnel, and the precipitate on the filter is washed with acetonitrile. The filtrate is concentrated to about 120 mL under reduced pressure. Water (120 mL) is added and the mixture is stirred for 40 minutes at 45 ° C and then cooled to ambient temperature. After 30 min, the mixture was filtered through a fried funnel to give 3 - ((trimethylsilyl) ethynyl) -10, 1-dihydro-5Hdibenzo [c, g] Roman-8 (9H) -one (4.07 g , with a yield of 33.4%), as a yellow solid: 400 MHz Ή NMR (CDC1₃) δ 7.65 (d, J = 8.1 Hz, 1H), 7.60 (s, 1H), 7.55 (s, 1H), 7.47 (dd, 7 = 8.1, 1, 4 Hz, 1H), 7.27 (s, 1H), 5.06 (s, 2H), 2.95 (t, J = 6.1 Hz, 2H), 2.67 - 2.59 (m, 2H), 2.18-2.08 (m, 2H), 0.26 (s, 9H).

3-acetyl-10, ll-dihydro-5H-dibenzo [c, g] chromen-8 (9H) -one

A 20 mL flask with a magnetic stirring stick is charged with 3 ((trimethylsilyl) ethynyl) -10, l-dihydro-5H-dibenzo [c, g] chromo-8 (9H) -one (850 mg, 2 , 44 mmol) and formic acid (9.8 mL). The solution is heated to 65 ° C. After 3 hours, the reaction is considered complete. The mixture is concentrated under reduced pressure; the resulting residue is taken up in CH₂C1₂ and is loaded onto a cartridge pre-packed with 25 g of silica gel. The product is purified by chromatography on a cartridge pre-packed with 80 g of silica gel eluting with a solvent gradient from 5% to 85% EtOAc / hexanes. The fractions, containing the product, were combined and concentrated to afford 3-acetyl-10,11-dihydro-5H-dibenzo [c, g] chromen-8 (9H) -one (616 mg, 86%): 400 MHz^!H NMR (CDC1)₃) δ 8.00 - 7.94 (m, 1H), 7.81 (d, J = 8.2 Hz, 1H), 7.77 (s, 1H), 7.64 (s, 2H), 5 , 16 (s, 2H), 2.98 (t, J = 6.1 Hz, 2H), 2.69 - 2.64 (m, 2H), 2.63 (s, 3H), 2.21 - 2.09 (m, 2H).

9-bromo-3- (2-bromoacetyl) -10, ll-dihydro-5H-dibenzo [c, g] chromen-8 (9H) -one

A 20 mL flask with a magnetic stirring stick is charged with 3-acetyl10, ll-dihydro-5H-dibenzo [c, g] chromen-8 (9H) -one (100 mg, 0.366 mmol), 9: 1 CH₂Cl₂/ MeOH (3.4 mL) and pyridinium tribromide (246 mg, 0.769 mmol). The solution itself

MD 4403 Cl 2016.09.30 warms up to 5 ° C. After 30 minutes, the reaction is considered complete. The mixture is cooled to ambient temperature, diluted with EtOAc (50 mL) and washed successively with aqueous saturated Na solution₂S₂A₃ (20 mL), aqueous NaHCO solution₃ 2% (20 mL), water (20 mL) and brine (10 mL). The organic phase is dried over MgSO₄, filtered and concentrated under reduced pressure resulting in 9-bromo-3 (2-bromacetyl) -10, 1-dihydro-5H-dibenzo [c, g] chromo-8 (9H) -one (68 mg, 41 %): 400 MHz 'H NMR (CDC1₃) δ 8.03 - 8.01 (m, 1H), 7.85 (d, J = 8.2 Hz, 1H), 7.82 (s, 1H), 7.71 (s, 1H), 7 , 67 (s, 1H), 5.19 (s, 2H), 4.74 (dd, J = 4.1, 4.1 Hz, 1H), 4.45 (s, 2H), 3.37 - 3.29 (m, 1H), 2.99 - 2.92 (m, 1H), 2.59 - 2.46 (m, 2H).

Example PY-4

<img file="MD4403C1_D0105.tif" />

<img file="MD4403C1_D0106.tif" />

Bcid (2S, 5S) * Î- (tert-butoxicaFb'Qnil) 5-methylpyr! Idin-2-carbc&gt; xih £ bramoacetiJF WJ1-dihsdro-5Hl · dibH &amp; nzofcgJcromen8 (9Н) -опа

<img file="MD4403C1_D0107.tif" />

{25Д5} -2 - {2- (9-bro mo-8- dxd-8, 9, ΐ 0 = ϊ 1 -tetra h id rc-5R-d ibenzof c, g] c roman-3-H) 2 5-methyl tert-butyl tert -butyl butyl 1,2-dicarboxylic acid (2S, 5S) -2- (2- (9-bromo-8-oxo-8.9, 10, II-tetrahydro-5H-dibenzo [c, g] chromen-3-yl) 2-oxoethyl) 1-tert-butyl 5-methylpyrrolidine-1,2-dicarboxylate

9-bromo-3- (2-bromacetyl) -10,11-dihydro-5H-dibenzo [c, g] chromen-8 (9H) -one (1.43 g, 3.17 mmol) is treated with о solution (2S, 5S) -l- (tert-butoxycarbonyl) -5-methylpyrrolidine-2-carboxylic acid (800 mg, 3.49 mmol) in dichloromethane (14 mL) and K₂CO₃(658 mg, 1.18 mmol). The stirred reaction mixture was stirred at room temperature and diluted with CH₂C1₂ and extracted 3 times. The organic phase was washed with brine, then dried over MgSO₄, filter and concentrate under reduced pressure to obtain ((2S, 5S) -2- (2- (9-bromo-8-oxo-8, 9,10, II-tetrahydro-5H-dibenzo [c, g ] chromen-3-yl) -2-oxoethyl) -1'-butyl-5-methylpyrrolidine-1,2-dicarboxylate (1.61 g, 84%).

This synthesis can be used to prepare a variety of compounds described herein, including the compound exemplified in PY.

BIOLOGICAL TESTS

Effect of serum protein on the potency of replicon: replication assays are performed in a normal cell culture medium (DMEM + 10% FBS) supplemented with physiological concentrations of human serum albumin (40 mg / mL) or acid glycoprotein (1 mg / mL). . EC₅₀ The presence of human serum proteins is compared with EC₅₀ in normal environment to determine the migration in the direction of potency.

MT-4 cell cytotoxicity: MT4 cells are treated with serial dilutions of compounds for a period of five days. Cell viability is measured at the end of the treatment period using the Promega CellTiter-Glo test, and nonlinear regression is performed to calculate CC₅oConcentration of the compound associated with the cells at EC₅₀: Huh-luc cultures are incubated with the compound at concentrations equal to EC 50. At several time intervals (072 hours), the cells are washed twice with cold medium and extracted with 85% acetonitrile; also, the sample of media at each time interval will be extracted. Extracts from cells and media are analyzed by LC / MS / MS to determine the molar concentration of the compounds in each fraction. The representative compounds of the invention showed activity.

Solubility and stability: Solubility is determined by taking an aliquot of 10 mM DMSO stock solution and preparing the compound at 100 µm fmal concentrations in test media solutions (PBS, pH 7.4 and 0.1Ν HC1, pH 1.5 ) with a total concentration of DMSO of 1%. The test media solutions are incubated at

MD 4403 Cl 2016.09.30 room temperature, with stirring for 1 hour. Then the solutions are centrifuged and the recovered supernatants are analyzed by HPLC / UV. The solubility is calculated by comparing the quantity of the detected compound in the defective test solution compared with the amount detected in DMSO at the same concentration. The stability of the compounds after 1 hour incubation with PBS at 37 ° C will also be determined.

Stability in cryopreserved hepatocytes of humans, dogs and rats: Each compound is incubated for up to 1 hour in hepatocyte suspensions (100 µL, 80,000 ° cells per alveolus) at 37 ° C. Cryopreserved hepatocytes are reconstituted in the serum-free incubation medium. The suspension is transferred to 96-well plates (50 µL / well). The compounds are diluted to 2 μΜ in the incubation medium and then added to the hepatocyte suspensions to begin incubation. The samples are taken at 0, 10, 30 and 60 minutes after the start of incubation and the reaction is quenched with a mixture of 0.3% formic acid in 90% acetonitrile / 10% water. The concentration of the compound in each sample is analyzed by LC / MS / MS. The decomposition period of the compound in the hepatocyte suspension is determined by the selection of the concentration-time data by a single-phase exponential equation. The data will also be extended to represent intrinsic liver clearance and / or total liver clearance.

Stability in hepatic S9 fraction in humans, dogs and rats: Each compound is incubated for up to 1 hour in S9 suspension (500 µL, 3 mg protein / mL) at 37 ° C (n = 3). The compounds are added to the S9 suspension to begin incubation. Samples are taken at 0, 10, 30 and 60 minutes after incubation begins. The concentration of the compound in each sample is analyzed by LC / MS / MS. The decomposition period of compound m S9 suspension is determined by the selection of concentration-time data by a single-phase exponential equation.

Caco-2 Permeability: Compounds are analyzed through a contract service (Absorption Systems, Exton, PA). The compounds are fumizaphs to the contractor in a blinded manner. Both direct permeability (A-to-B) and reverse permeability (В-to-A) will be measured. Caco-2 monolayers are grown at confluence on collagen-coated microporous polycarbonate membranes in Costar TRANS WELL® boxes with 12 alveoli. The compounds are dosages on the apical side for direct permeability (A-to-B) and are dosages on the basolateral side for inverse permeability (B-to-A). The cells are incubated at 37 ° C with 5% CO₂ in a humidified incubator. At the beginning of incubation and at 1 hour and 2 hours after incubation, an aliquot of 200 µL is taken from the reception room and replaced with a fresh test buffer. The concentration of the compound in each sample is determined by LC / MS / MS. The apparent permeability of Papp is calculated.

Plasma protein binding: Plasma protein binding is measured by balanced dialysis. Each compound is enriched in an immaculate plasma at о final concentration of 2 μΜ. The enriched plasma and phosphate buffer are placed in the opposite ends of the assembled dialysis cells, which are then slowly rotated in a water bath at 37 ° C. At the end of the incubation, the concentration of the compound in plasma and phosphate buffer is determined. The unrelated percentage is calculated using the following equation:

% Unrelated = 100 ·

C_b+ Cf where Cf and C_b are the free and bound concentrations determined as the concentrations of plasma and post-dialysis buffer, respectively.

CYP450 profiling: Each compound is incubated with each of the 5 recombinant human CYP450 enzymes, including CYP1A2, CYP2C9, CYP3A4, CYP2D6 and CYP2C19 in the presence and absence of NADPH. Serial samples will be taken from the incubation mixture at the beginning of the incubation and at 5, 15, 30, 45 and 60 minutes after the beginning of the incubation. The concentration of the compound in the incubation mixture is determined by LC / MS / MS. The percentage of the compound remaining after incubation at each time interval is calculated by comparison with the sample at the beginning of incubation.

Plasma stability in humans, dogs, monkeys and rats: The compounds will incubate for up to 2 hours in plasma (rats, dogs, monkeys or humans) at 37 ° C. The compounds are added to the plasma in final concentrations of 1 and 10 pg / mL. The aliquots are taken at 0, 5, 15, 30, 60 and 120 min after adding the compound.

MD 4403 Cl 2016.09.30

Concentration of compounds and main metabolites! at each time interval is measured by LC / MS / MS.

Evaluation of cellular anti-HCV activity: Antiviral potency (EC₅₀) was determined using a HCV replicon reporter assay based on (RLuc) Renilla luciferase. To perform the JFH-1 genotype 1 and 2a assay, HCV cells in stable replicating RLuc (harboring a H77 replicon of the dicistronic genotype encoding an RLuc reporter), stable replicating HCV lb RLuc cells (harboring a cone-replicon lb of the dicistronic genotype which encodes an RLuc reporter) or stable replicating VHC 2a JFH-1 Rluc cells (housing a JFH-1 replicon 2a of the dicistronic genotype encoding an RLuc reporter; with L31 present in NS5A) were distributed in boxes with 384 alveoli for EC tests₅₀. To perform the test for genotype 2a (with M31 present in NS5A) or 2b, replicons 2a JFH-1 chimeric genotype NS5A encoding an RLuc-Neo reporter and either genotype 2a J6 strain NS5A strain or genotype NS5A strain strain 2b MD2b-l ( both with M31 present), respectively, were either temporarily transfected (t) into Huh-Lunet cells or established as stable replication replicon cells). Either the cells were distributed in boxes with 384 cells for EC tests₅₀. To perform the assay for genotype 3 and 4, the chimeric NS5A genotype NS5A replication codons encoding a Pi-RLuc reporter and either the genotype 3a strain S52 of the NS5A gene or the genotype 4a ED43 of the strain of the NS5A gene, were temporally transfected (t) into Huh cells. - Lunet, which were subsequently distributed in boxes with 384 alveoli. The compounds are dissolved in DMSO, at о 10 mM concentrations and diluted in DMSO either manually or by means of an automated drip tool. The 3-fold diluted compounds are either manually mixed with cell culture media and added to seed cells or added directly to cells using an automated tool. DMSO is used as a negative control (solvent; no inhibition), and the ITMN-191 protease inhibitor is included in a concentration&gt; 100 x EC₅or as a positive control. 72 hours later, cells were lysed and Renilla luciferase activity quantified as recommended by the manufacturer (PromegaMadison, WI). Nonlinear regression was performed to calculate EC values₅₀.

To determine antiviral potency (EC₅₀) against resistant mutants, resistance mutations, including M28T, Q30R, Q30H, L31M and Y93C in the genotype at NS5A and Y93H in the genotype lb NS5A, were introduced individually either in the replicons to PiRluc or lb Pi-Rluc by site mutagenesis. specific. The RNA replicon of each resistant mutant was transfected into treated cells-51 derived from Huh-7 and antiviral potency was determined on these transfected cells as described above.

The EC50 for genotypes at, at Q30R and 2a JFH are as follows: A&gt; 44 nM, В = 1 nM at 43.99 nM, с &lt;1 nM. The frets ЕС₅₀ for genotype 2a J6, 2b, 3a and 4a are as follows: A&gt; 5 nM, В = 1 nM at 4.99 nM, с &lt;1 nM. The frets ЕС₅₀ for genotype 2a J6, 2b and 4a correspond to the analysis of the transfected cells (t) temporarily. If this data is not available, the EC50 slide for stable replication cell (s) is applied.

Pharmacokinetic studies of single dose IV and PO in SD rats: The pharmacokinetics of the selected compounds were characterized in male SpragueDawley (SD) rats (250-3OOg). In this study, two groups of naive rats of SD (N = 3 per group, overnight stays) received the selected compound either as intravenous (IV) infusion (1 mg / kg for 30 minutes) through the jugular vein or via artificial oral diet (2 mg / kg). The intravenous (IV) carrier was 5% ethanol, 35% polyethylene glycol 400 (PEG 400) and 60% water pH 2.0. The oral carrier was 5% ethanol, 55% PEG 400 and 40% citrate buffer pH 2.2.

Serial blood samples (approximately 0.3 mL each) were collected from the jugular vein or other suitable vein at specified time intervals. For group IV infusion, blood samples were collected before dosing and at 0.25, 0.48, 0.58, 0.75, 1.5, 3, 6, 8, 12 and 24 hours after the start of infusion. . For the oral group, blood samples were collected before dosing and at 0.25, 0.50, 1, 2, 4, 6, 8, 12 and 24 hours after dosing. Blood samples were collected in Vacutainer ™ tubes containing EDTA-K3 as an anticoagulant and centrifuged at approximately 4 ° C to obtain plasma. Plasma samples were stored at -20 ° C until analyzed by LC / MS / MS.

The bioanalytical method using high performance liquid chromatography coupled with tandem mass spectrometry (LC / MS / MS) has been developed

MD 4403 Cl 2016.09.30 for the analysis of the selected compound from rat plasma. Detection was performed using selected reaction monitoring (MRS); lanes representing precursor species (M + H)⁺ were selected in quadripole 1 (QI) and collided with argon gas in the collision cell (Q2) to generate specifically produced ions, which were subsequently monitored by quadripole 3 (Q3). Quality control samples and standard curve were prepared in male rat plasma and processed in the same way as the test samples to generate quantitative data.

Pharmacokinetic parameters were generated using non-compartmental pharmacokinetic analysis (Phoenix WinNonlin, version 6.3). Values ​​below the lower limit of quantification (LIC) were assigned a value of zero for the preliminary dose and were subsequently treated as missing. The area under the curve (AUC) was calculated by the linear trapezoidal rule. Oral bioavailability (F%) was determined by comparing the area under the curve (AUC) of the compound and / or a metabolite generated in plasma after oral administration with that generated after intravenous administration.

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MD 4403 Cl 2016.09.30 (56) Bibliographic references cited in the description:

1. Antimicrobial Agents and Chemotherapy, September 2010, Volume 54, pp. 3641-3650

2. Paquette, Leo A.; Principles of Modern Heterocyclic Chemistry (WA Benjamin, New York, 1968), in particular Chapters 1, 3, 4, 6, 7 and 9;

3. The Chemistry of Heterocyclic Compounds, A Series of Monographs ”(John Wiley &amp; Sons, New York, 1950 to date), especially volumes 13, 14, 16, 19 and 28;

4. J. Am. Chem. Shock. (1960) 82: 5566

5. SP Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984), McGraw-Hill Book Company, New York;

6. Eliel, E. and Wilen, S., Stereochemistry of Organic Compounds (1994) John Wiley &amp; Sons, Inc., New York

7. Bundgaard, Hans, "Design and Application of Prodrugs" in Textbook of Drug Design and Development (1991),

8. P. Krogsgaard-Larscn and H. Bundgaard, Eds. Harwood Academic Publishers, pp.113-191

9. Farquhar, (1983) J. Pharm. Sci. 72: 324;

10. US 4816570,

11. US 4968788,

12. US 5663159

13. US 5792756

14. De Lombaert et al. (1994) J. Med Chem. 37: 498)

15. Khamnei and Torrence, (1996) J. Med. Chem. 39: 4109-4115.

16. Mitchell et al., (1992) J. Chem. Shock. Perkin Trans. II2345;

17. Glazier WO 91/19721.

18. Puech et al. (1993) Antiviral Res., 22: 155-174;

19. Benzaria et al. (1996) J. Med. Chem. 39: 4958)

20. Protective Groups in Organic Chemistry, Theodora W. Greene, John Wiley &amp; Sons, Inc., New York, 1991

21. Protective Groups in Organic Synthesis, Theodora W. Greene (John Wiley &amp; Sons, Inc., New York, 1991, ISBN 0-471-62301-6) ("Greene")

22. Kocienski, Philip J.; Protecting Groups (Georg Thieme Verlag Stuttgart, New York, 1994)

2. 3. Chapter 1, Protecting Groups: An Overview, pages 1-20,

24. Chapter 2, Hydroxyl Protecting Groups, pages 21-94,

25. Chapter 3, Diol Protecting Groups, pages 95-117,

26. Chapter 4, Carboxyl Protecting Groups, pages 118 - 154,

27. Chapter 5, Carbonyl Protecting Groups, pages 155-184

28. Handbook of Pharmaceutical Excipients (1986)

29. Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton, PA)

30. Compendium of Organic Synthetic Methods (John Wiley &amp; Sons, New York), Vol. 1, Ian T. Harrison and Shuyen Harrison, 1971;

31. Vol. 2, Ian T. Harrison and Shuyen Harrison, 1974,

32. Vol. 3, Louis S. Hegedus and Leroy Wade, 1977,

33. Vol. 4, Leroy G. Wade, Jr., 1980,

34. Vol. 5, Leroy G. Wade, Jr., 1984;

35. and Vol. 6, Michael B. Smith;

36. March, J., Advanced Organic Chemistry, Third Edition, (John Wiley &amp; Sons, New York, 1985),

37. Comprehensive Organic Synthesis. Selectivity, Strategy &amp; Efficiency in Modern Organic Chemistry. In 9 volumes, Barry M. Trost, Senior Editor (Pergamon Press, New York, 1993 edition)

38. WO 2006/020276

MD 4403 Cl 2016.09.30

39. (Stereochemistry of Carbon Compounds, (1962) by EL Eliel, McGraw Hill; Lochmuller, CH, (1975) /. Chromatogr., 113, 3) 283-302)

40. Eliel, E. and Wilen, S. (1994) Stereochemistry of Organic Compounds, John Wiley &amp; Sons, Inc., p. 322

41. Jacob III (. 1982) J. Org. Chem. 47: 4165)

42. Hoye, T., WO 96/15111

43. Chiral Liquid Chromatography (1989) WJ Lough, Ed. Chapman and Hall, New York; Okamoto, (1990) /. of Chromatogr. 513: 375-378)

44. Wuts, PGM, Greene, T. Protective Groups in Organic Synthesis, 4th edition;