EA001316B1 - Anti-viral compounds - Google Patents

Abstract

1. A compound of the formula I wherein: a is 0, 1, 2 or 3; each R is independently hydrogen, halo, cyano, amino, halo(C1-C6)alkyl, di(C1-C4)alkylamino, azido, C1-C6 alkyl, carbamoyl, carbamoyloxy, carbamoylamino, C1-C6 alkoxy, C1-C4 alkylthio, C1-C4 alkylsulfinyl, C1-C4 alkylsulfonyl, pyrrolidino, piperidino or morpholino; R<0> is hydrogen, halo, C1-C4 alkyl or C1-C4 alkoxy; R<1> is halo, cyano, hydroxy, methyl, ethyl, methoxy, ethoxy, methylthio, methylsulfinyl or methylsulfonyl; R<2> is hydrogen, amino or -NHC(O)( C1-C6 alkyl); R<3> is dimethylamino, C1-C10 alkyl, C3-C7 cycloalkyl, substituted C3-C7 cycloalkyl, halo(C1-C6)alkyl, phenyl, substituted phenyl, furyl, thienyl, thiazolyl, thiazolidinyl, pyrrolidino, piperidino, morpholino or a group of the formula R<4> and R<5> are independently hydrogen or C1-C4 alkyl; or a pharmaceutically acceptable salt thereof. 2. A compound according to claim 1 where: a is 0, 1 or 2; each R is independently hydrogen, halo, C1-C4 alkyl, C1-C4 alkoxy or di(C1-C4)alkylamino; R<0> is hydrogen; R<2> is amino; R<3> is dimethylamino, C1-C6 alkyl, halo(C1-C6)alkyl, phenyl, substituted phenyl, C3-C7 cycloalkyl, substituted C3-C7 cycloalkyl, thienyl, thiazolidinyl, pyrrolidino, piperidino or morpholino; R<4> is hydrogen, methyl or ethyl; R<5> is hydrogen, methyl or ethyl; or a pharmaceutically acceptable salt thereof. 3. A compound according to claim 2 where: a is 0 or 1; each R is independently hydrogen, fluoro, methyl, ethyl, methoxy, ethoxy, dimethylamino; R<3> is C1-C4 alkyl, phenyl, substituted phenyl, C3-C7 cycloalkyl or substituted C3-C7 cycloalkyl; or a pharmaceutically acceptable salt thereof. 4. A compound according to claim 3 which is or a pharmaceutically acceptable salt thereof. 5. A pharmaceutical formulation comprising a compound of formula I, or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 4, associated with one or more pharmaceutically acceptable carriers, diluents or excipients. 6. A compound of formula I, or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 4, for use as a pharmaceutical. 7. A compound as claimed in claim 6, for use as an antiviral.

Description

This invention relates to medicine, in particular to the treatment of viral infections. More specifically, this invention relates to the treatment of rhinovirus, enterovirus and flavivirus infections.

The prevalence of upper respiratory tract disease, the common cold, is huge. It is estimated that nearly a billion cases per year occur only in the United States. Rhinovirus, a member of the Ryogatttbae family. is the main causative agent of the common cold in humans. Due to the fact that more than 110 strains of rhinoviruses have been identified, the development of a practical rhinovirus vaccine is not feasible, and chemotherapy seems to be a more desirable approach to fight this type of infection. Another member of the P1cogpaushbay family is enterovirus, which includes approximately eighty human pathogens. Many of these viruses cause cold-like symptoms; others cause more serious illnesses, such as polio, conjunctivitis, aseptic meningitis, and myocarditis.

A disease associated with rhinovirus infection has symptoms such as nasal discharge and nasal obstruction. In addition, it was believed that it was related to inflammation of the middle ear, provokes the development of bronchitis, exacerbates sinusitis and is related to precipitation of asthmatic aIosyk. Although many consider this disease to be just an annoying annoyance, its frequent occurrence in otherwise healthy individuals and the resulting economic importance in the form of a prolonged absence of employees and doctor's visits make this disease the subject of extensive research.

The ability of chemical compounds to suppress the growth of ίη νίίτο viruses can be easily demonstrated using the suppression test of viral plaques or the test of cytopathogenic effect (CPE). See ΞίιηίηοΓΓ. Arrbeb Mutiuyuyuuu 9 (1). 66 (1961). Although a number of chemical compounds that inhibit picornaviruses, such as rhinoviruses, have been identified, many are unacceptable due to 1) a limited spectrum of activity, 2) undesirable side effects, or 3) inability to prevent infection or illness in animals or humans. See TechLooc oG Nitap UlooGy. ebayeb vu voej V. Vekye. syar1et 16. Prypo zgikek. V.A. ke \ ^r apbo \\ '5k |. 391-405 (1985). Thus, despite the recognized therapeutic potential associated with the rhinovirus inhibitor and the research attempts undertaken so far, a viable therapeutic agent has not yet been identified. For example, antiviral derivatives of benzimidazole have been described in and. 8. Ra1.

8th Hawk 4 008 243. 4 018 790. 4 118 573. 4 118 742. 4 174 454 and 4 492 708.

In General, the compounds described in the above patents do not have the desired pharmacological profile for use in the treatment of rhinovirus infections. Specifically, these compounds do not have satisfactory oral bioavailability or a sufficiently high inhibitory activity to compensate for their relatively low oral bioavailability in order to allow their widespread use. In addition, it is widely recognized in the art that the compounds used to treat rhinovirus infections should be non-toxic.

Therefore, the object of the present invention is to provide novel benzimidazole compounds that inhibit the growth of picornaviruses, such as rhinoviruses, enteroviruses, such as polioviruses, coxsiviruses of groups A and B, or ECHO virus, and which have the desired pharmacological profile.

This invention relates to compounds of formula I:

o = s

I.

NB ⁴ K ⁵ where a is 0, 1. 2 or 3;

each B is independently hydrogen, halogen, cyano, amino, halo (C ₁ -C ₄₎ alkyl, di (C1-C4) alkylamino, azido, C1-C6 alkyl, carbamoyl, carbamoyloxy, carbamoylamino, _C1-C4 alkoxy , _C1-C4 alkylthio, _C1-C4 -alkilsulfinil, C1-C4 alkylsulfonyl, pyrrolidino, piperidino or morpholino;

At ⁰ is hydrogen, halogen, C ₁ -C ₄ alkyl or C ₁ -C ₄ alkoxy;

B ¹ is halogen, cyano, hydroxy, methyl, ethyl, methoxy, ethoxy, methylthio, methylsulfinyl or methylsulfonyl;

B ² is hydrogen, amino or -1XNS (O) (C! -C ₆ alkyl);

B ³ is dimethylamino, C1-C10 alkyl, C ₃ -C ₇ alkyl, substituted C ₃ -C ₇ cycloalkyl, halogen (C ₁ -C ₆ ) alkyl, phenyl, substituted phenyl, furyl, thienyl, thiazolidinyl, pyrrolidino , piperidino, morpholino, thiazolyl or a group of the formula ξ A:. M '

B ⁴ and B ⁵ independently represent hydrogen or C1-C4 alkyl;

or a pharmaceutically acceptable salt thereof.

The invention also relates to a pharmaceutical composition comprising a compound of the invention, or a pharmaceutically acceptable salt thereof, in combination with a pharmaceutically acceptable carrier, diluent or excipient.

The present invention also relates to a method for inhibiting picornavirus, comprising administering to a host in need of treatment an effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof, wherein a, K, K ⁰ , K ¹ , K ² , K ³ , K ⁴ and K ⁵ are as defined above.

The present invention also relates to a method of inhibiting flavivirus, comprising administering to a host in need of treatment an effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof, wherein a, K, K ⁰ , K ¹ , K ² , K ³ , K ⁴ and K ⁵ are as defined above.

All temperatures given here are in degrees Celsius (° C). All units used here are given in weight units, except liquids, for which volume units are given.

As used herein, the term C ₁ -C ₁₀ alkyl means a straight or branched alkyl chain having from one to ten carbon atoms. Typical C ₁ -C ₁₀ alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, neopentyl, hexyl, 2-methylhexyl, heptyl and the like. The term C ₁ -C ₁₀ alkyl includes in its definition the terms C ₁ -C ₆ alkyl and C ₁ -C ₄ alkyl.

Halogen means chlorine, fluorine, bromine or iodine.

Halogen (C ₁ -C ₄ ) alkyl denotes a straight or branched alkyl chain having from one to four carbon atoms, with 1, 2 or 3 halogen atoms attached to it. Typical halogen (C ₁ -C ₆ ) alkyl groups include chloromethyl, 2-bromoethyl, 1-chloroisopropyl, 3-fluoropropyl, 3-bromobutyl, 3chlorisobutyl, iodo-tert-butyl, trichloromethyl, trifluoromethyl, 2,2-chloroethyl, 2, 3dibromopropyl, etc.

C ₁ -C ₄ alkylthio denotes a straight or branched alkyl chain having from one to four carbon atoms attached to a sulfur atom. Typical C ₁ -C ₄ alkylthio groups include methylthio, ethylthio, propylthio, isopropylthio, butylthio and the like.

C ₁ -C ₆ alkoxy denotes a straight or branched alkyl chain having from one to six carbon atoms attached to an oxygen atom. Typical C ₁ -C ₆ alkoxy groups include methoxy, ethoxy, propoxy, isopropoxy, butoxy, and the like. The term C _{1 to} C ₆ alkyl includes within its definition the term C ₁ -C ₄ alkyl.

Di (C ₁ -C ₄ ) alkylamino refers to two straight or branched alkyl chains having from one to four carbon atoms attached to a common amino group. Typical di (C1-C4) alkylamino groups include dimethylamino, ethylmethylamino, methylpropylamino, ethylisopropylamino, butylmethylamino, sec-butyl ethylamino, etc.

C ₁ -C ₄ -alkylsulfinyl is a straight or branched alkyl chain having from one to four carbon atoms attached to a sulfinyl radical. Typical C ₁ -C ₄ alkylsulfinyl groups include methylsulfinyl, ethylsulfinyl, propylsulfinyl, isopropylsulfinyl, butylsulfinyl, etc.

C1-C4-alkylsulfonyl means a straight or branched alkyl chain having from one to four carbon atoms attached to a sulfonyl radical. Typical C1-C4 alkylsulfonyl groups include methylsulfonyl, ethylsulfonyl, propylsulfonyl, isopropylsulfonyl, butylsulfonyl and the like.

Substituted phenyl means a phenyl ring substituted with one to three substituents selected from the following substituents:

halogen, cyano, C ₁ -C ₄ alkyl, C ₁ -C ₄ alkoxy, amino or halogen (C ₁ -C ₄ ) alkyl.

Substituted C ₃ -C ₇ cycloalkyl means a cycloalkyl ring substituted with one to three substituents selected from the following substituents: halogen, cyano, C ₁ -C ₄ alkyl, C ₁ -C ₄ alkoxy, amino or halogen (C ₁ C 4) alkyl.

The claimed compounds can occur either in cis or trans conformation. For the purposes of this invention, cis - refers to compounds in which the carboxamide moiety is in the cis position relative to the benzimidazole ring, and trans - refers to compounds in which the carboxamide moiety is in the trans position relative to the benzimidazole ring. Both isomers are included in the scope of the claimed compounds.

As mentioned above, this invention includes pharmaceutically acceptable salts of the compounds of formula I. The compound of this invention may possess sufficiently acidic, sufficiently basic, or both functional groups and, accordingly, interact with any of a number of inorganic bases and inorganic and organic acids to form a pharmaceutically acceptable salt.

The term pharmaceutically acceptable salt, as used here, refers to salts of the compounds of the above formula, which are essentially non-toxic to living organisms. Representative pharmaceutically acceptable salts include those prepared by reacting the compounds of this invention with a mineral or organic acid or inorganic base. Such salts are known as acid addition salts and base addition salts.

The acids commonly used to form acid addition salts are inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid and the like, and organic acids such as p-toluenesulfonic acid, methanesulfonic acid , ethanesulfonic acid, oxalic acid, p-bromophenyl sulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, etc.

Examples of such pharmaceutically acceptable salts are sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, heptate, isobutyl, isobutate, isobutate, isobutate, isobutate, isobutate propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyn-1,4-dioate, hexin-1,6-dioate, benzoate, chlorobenzoate, methyl benzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, sulfonate, phthalate, phenylacetate, phenylpropion t, phenyl butyrate, citrate, lactate, γ-hydroxybutyrate, glycolate, tartrate, methanesulfonate, ethanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate, and the like. Preferred pharmaceutically acceptable acid addition salts are those formed with mineral acids such as hydrochloric acid and sulfuric acid and those formed with organic acids such as maleic acid and methanesulfonic acid.

Base addition salts include salts formed from inorganic bases such as ammonium or alkali or alkaline earth metal hydroxides, carbonates, bicarbonates, and the like. Such bases used in the preparation of the salts of the present invention include, therefore, sodium hydroxide, potassium hydroxide, ammonium hydroxide, sodium carbonate, sodium bicarbonate, potassium bicarbonate, calcium hydroxide, calcium carbonate and the like. Forms of potassium and sodium salts are particularly preferred.

It should be understood that the nature of the particular counterion forming part of any salt of the present invention is not critical as long as the salt is generally pharmaceutically acceptable and as long as this counterion does not add the undesirable qualities of the salt as a whole.

Preferred compounds of this invention are compounds

o = s

NK ⁴ K ⁵ where a is 0, 1 or 2;

each K is independently hydrogen, halogen, C ₁ -C ₄ alkyl, C ₁ -C ₄ alkoxy or di (C ₁ C ₄ ) alkylamino;

K ⁰ is hydrogen;

K ² is amino;

K ³ is dimethylamino, C ^ C ₆ -alkyl, halogen (C ₁ -C ₆ ) alkyl, phenyl, substituted phenyl, C ₃ -C ₇ -cycloalkyl, substituted C ₃ -C ₇ cycloalkyl, thienyl, thiazolidinyl, pyrrolidino, piperidino or morpholino;

K ⁴ is hydrogen, methyl or ethyl;

K ⁵ is hydrogen, methyl or ethyl; or a pharmaceutically acceptable salt thereof.

Of these preferred compounds, compounds of formula I are more preferred, wherein a is 0 or 1;

each K is independently hydrogen, fluoro, methyl, ethyl, methoxy, ethoxy, dimethylamino;

K ³ is C 1 -C 4 alkyl, phenyl, substituted phenyl, C ³ -C ⁷ cycloalkyl or substituted C ³ -C ⁷ cycloalkyl;

or a pharmaceutically acceptable salt thereof.

Of these compounds, the most preferred compounds are

or a pharmaceutically acceptable salt thereof.

Compounds of formula I can be prepared by reacting a suitably substituted acetamide with a base to produce the corresponding anion, which then reacts with a suitably substituted ketone of formula ΙΑ to form a carbinol intermediate. These reactions are usually carried out in an organic solvent for one to twelve hours at a temperature of from about -90 ° C to room temperature using an excess of base and acetamide reagent relative to the ketone reagent. Before use in this reaction, acetamide is preferably protected with a suitable protecting group. Typical bases include sodium hydride, lithium diisopropylamide (LOL), and nbutyl lithium. A preferred base is n-butyllithium. The choice of solvent is not critical, as long as the solvent used is inert and the reagents are sufficiently solubilized to carry out the desired reaction. A solvent suitable for use in this reaction is tetrahydrofuran, although an acetamide reagent can also be used as a solvent. The carbinol intermediate is usually obtained within ~ 1 to 18 hours when the reaction is initiated at -78 ° C and allowed to slowly warm to room temperature. The reaction can be monitored by HPLC and quenched by the addition of acid when it is substantially complete. Typical acids include hydrochloric acid, hydrobromic acid, formic acid, and the like. Preferred acid is concentrated hydrochloric acid. The resulting carbinol intermediate is preferably dehydrated without prior isolation or purification.

In particular, the carbinol intermediate reacts with an acid for 30 minutes to 12 hours at a temperature from ~ room temperature to the reflux temperature (when heated under reflux) of the mixture to form the target compound of formula I. Typical acids include hydrochloric acid, hydrobromic acid, formic acid acid, acetic acid and combinations of acids. A preferred combination of acids is formic acid containing concentrated hydrochloric acid. The target compound is usually obtained over a period of time from ~ 30 minutes to 7 hours, when the reaction is carried out at a temperature slightly below the reflux temperature of the mixture. The reaction is preferably monitored by HPLC, for example, to ensure that the reaction is complete.

The compounds of formula I are preferably isolated and the resulting cis / trans isomers are separated using procedures known in the art. For example, the cis and trans forms of the isolated compounds can be separated by column chromatography, for example, reverse phase HPLC. Compounds can be eluted from the column using an appropriate ratio of acetonitrile to water or methanol to water. The cis form of the compound can be converted to a cis / trans mixture by exposure to radiation and recyclized through the aforementioned purification process.

Ketone intermediates of formula ΙΑ can be prepared according to procedures detailed in the art. For example, ketone intermediates can be prepared according to the following Reaction Scheme I.

Where

X is cyano or —COOE, where B ′ is C ₁ -C ₄ alkyl;

X 'is halogen;

a, B, B ⁰ , B ¹ , B ² and B ³ have the above meanings.

The reaction scheme I above is carried out by carrying out reactions 1-4. Upon completion of the reaction, the intermediate compound, if desired, can be isolated by methods known in the art. For example, the compound can be crystallized and then collected by filtration, or the solvent in which the reaction was carried out can be removed by extraction, evaporation or decantation. The intermediate, if desired, can be further purified by conventional means, such as crystallization or chromatography on solid supports, such as silica gel or silica, before carrying out the next step of the reaction scheme.

The T1 reaction is first performed by exposing a suitably substituted halogen nitroaniline and a suitably substituted phenylacetonitrile or benzoate with a base in an organic solvent for 1-24 hours at a temperature of from about -10 ° C to about 40 ° C to obtain a ketone precursor. This reaction is usually carried out using equimolar ratios of the reactants in the presence of two equivalents of base. Typical bases include sodium hydride, potassium tert-butoxide, lithium diisopropylamide (BOA). A preferred base is potassium tert-butoxide. Examples of solvents suitable for use in this reaction include dimethylformamide, dimethylacetamide and the like. The choice of solvent is not critical if the solvent used is inert and the reagents are sufficiently solubilized to carry out the desired reaction. The ketone precursor is usually obtained within ~ 1 to 15 hours when the reaction is initiated at 0 ° C and allowed to proceed at room temperature. The ketone precursor is preferably oxidized in the same reaction mixture without prior isolation or purification.

In particular, the ketone precursor is reacted with an oxidizing agent for 30 minutes to 15 hours at a temperature of from about 0 ° C to about 30 ° C to obtain the corresponding ketone compound. Typical oxidizing agents include hydrogen peroxide, oxygen, and air. Oxygen and air are usually bubbled through the reaction mixture. A preferred oxidizing agent is hydrogen peroxide, preferably in the form of a 30% solution. Ketone is usually obtained within about 30 minutes to 5 hours when the reaction is carried out at a temperature between 0 ° C. and room temperature. The reaction is usually preferably monitored by, for example, TLC to ensure that the reaction has been completed.

In reaction 1.2, the nitro substituent on the ketone is reduced in accordance with procedures known in the art to give the corresponding diaminobenzophenone derivative. For example, a nitro substituent can be reduced by catalytic hydrogenation, for example, by reacting a ketone isolated from reaction 1.1 with hydrogen gas in ethanol or tetrahydrofuran and in the presence of a catalyst. The preferred catalyst is palladium on carbon or Raney nickel. The choice of solvent is not critical if the solvent used is inert and the nitroreagent is sufficiently solubilized to carry out the desired reaction. Hydrogen gas is usually used at a pressure of up to 60 psi (413685.45 Pa), preferably at or about 30 psi (206842.71 Pa). The reaction usually essentially ends after about 1-24 hours, when it is carried out at a temperature ranging from ~ 0 ° C to ~ 40 ° C. The reaction is preferably carried out at a temperature ranging from ~ 20 ° C to ~ 30 ° C for ~ 2-5 hours

In reaction 1.3, a compound isolated from reaction 1.2 is cyclized through a nitrile intermediate by reacting a benzophenone compound with bromine cyan in an alcohol solvent such as isopropanol. Usually this reaction is carried out at a temperature of from ~ 0 ° C to ~ 30 ° C. With complete dissolution of benzophenone, the resulting solution is combined with cyanogen bromide. Bromocyanin is usually added in the form of a solution (3-7M, for example, in acetonitrile). The reaction usually ends after 1-18 hours when the reaction mixture is stirred at room temperature. However, in some cases, the nitrile intermediate will precipitate from the reaction mixture. To obtain the desired ketone, this precipitate is isolated and then refluxed in an alcohol solvent such as isopropanol for 1-4 hours to obtain the target ketone compound of formula I.

Compounds of the formula

Where

X ', B ⁰ and B ³ have the given meanings; obtained by substituting a chlorine or fluorine substituent for a compound of the formula:

where Υ is chlorine or fluorine, provided that Υ cannot be chlorine when X 'is fluorine, the primary amine of the formula ΝΗ ₂ Β ³ , where B ³ is as defined above, in an organic solvent. This reaction is optionally carried out in the presence of an acid acceptor, such as potassium carbonate, or a large excess of the primary amine. Typical solvents include tetrahydrofuran, dimethylformamide, dimethylacetamide, etc. The reaction usually ends within 1-20 hours when it is carried out at a temperature of from ~ 20 ° C to ~ 80 ° C. The resulting alkylated halogenonitroaniline then reacts as described in Reaction Scheme I above.

Compounds of formula I, in which B ² denotes -ΝΗΟ (Ο) _(C1-C6 alkyl) may be prepared by acylation of the ketone intermediate of the formula I or of the corresponding compounds wherein B ² is an amino group according to procedures known in the art area. For example, the amine compound may be acylated with a suitable acyl halide, isocyanate or chloroformate, preferably in the presence of an acid acceptor such as a tertiary amine, preferably triethylamine. The preferred acylating agent is acetic anhydride. This reaction is usually carried out at a temperature of from about −20 ° C. to about 25 ° C. Typical solvents used in this reaction include ethers and chlorinated hydrocarbons, preferably diethyl ether, chloroform or methylene chloride. The amine reagent is usually used in equimolar proportions relative to the acylating reagent and preferably in the presence of equimolar amounts of an acid acceptor such as a tertiary amine. A preferred acid acceptor for this reaction is Ν-methylmorpholine (ΝΜΜ).

The compounds used as starting materials in the synthesis of the compounds of this invention are well known in the art and, if they are not commercially available, can be easily synthesized by standard procedures commonly used in the art.

It should be clear to specialists in this field that when carrying out the above processes, it may be desirable to introduce chemical protective groups into the reactants to prevent the occurrence of adverse reactions. Any amino, alcohol, alkylamino, or carboxy groups that may be present on the reactants can be protected using a standard amino, hydroxy, or carboxy protecting group that does not adversely affect the ability of the rest of the molecule to react in a desired manner. A variety of protecting groups can then be removed simultaneously or sequentially using methods known in the art.

The pharmaceutically acceptable salts of this invention are usually formed by the reaction of a compound of formula I with an equimolar or excess acid or base. The reactants are usually combined in a common solvent, such as diethyl ether, tetrahydrofuran, methanol, ethanol, isopropanol, benzene and the like, for acid addition salts, or water, alcohol or a chlorinated solvent, such as methylene chloride, for base addition salts . Salts usually precipitate from solution within about one hour to about 10 days and can be isolated by filtration or other conventional methods.

The following preparations and examples further illustrate specific aspects of the present invention. However, it should be understood that these examples are included for illustrative purposes only and do not limit the scope of the invention in any way and should not be construed in this way.

In the following preparations and examples, the terms melting point, nuclear magnetic resonance spectra, electron impact mass spectra, field desorption mass spectra, accelerated atom bombardment mass spectra, infrared spectra, ultraviolet spectra, elemental analysis, high resolution liquid chromatography and thin layer chromatography has the abbreviations mp, ΝΜΚ (NMR), ΕΙΜ8 (EUMS), Μ8 (ΕΌ) (PDMS), M8 (EAB) (BCA)

MS), ΙΚ (IR), υν (UV), AIA1U818 (analysis), HPLC (HPLC) and TBC (TLC), respectively. Data Μ8 (ΕΌ) is presented as a mass number, unless otherwise indicated. In addition, the absorption maxima listed for the IR spectra are only maxima of interest, but these are not all observed maxima.

In connection with the NMR spectra, the following abbreviations were used: c stands for singlet, b stands for doublet, bb stands for doublet of doublets, 1 stands for triplet, s. | denotes a quartet, m denotes a multiplet, bt denotes a doublet of multiplets, and bt, bt, bt, 1 and bgt mean a wide singlet, doublet, triplet and multiplet, respectively. 1 indicates the interaction constant in Hertz (Ηζ). Unless otherwise indicated, NMR data refer to the free base of the test compound.

NMR spectra were obtained on a Vgikeg Sogr setup. 250 MHz or on the installation Oeyega1 E1ec1ps ΟΕ-300 300 MHz. Chemical shifts were expressed in delta, δ-values (millionths of a weak-field shift from tetramethylsilane). Μ8 (EE) spectra were recorded on a νηποη-ΜαΙ 731 spectrometer using carbon dendritic emitters. ΕΙΜδ spectra were obtained on a CEC 21-110 setup from Coi8o11ba1eb E1ec1tobupatyuv Sotrotiop. IR spectra were obtained on a Regk1p-E1teg 281 instrument. UV spectra were obtained on a Saga 118 instrument. TLC was performed on E. Μе ^ sk silica gel plates. Melting points are uncorrected.

Example 1. A. 2-Isopropylamino-4-fluoronitrobenzene.

To a cold (0 ° C.) mixture of 43, 35 ml (400 mmol) of 2,4-difluoronitrobenzene and 55 g (approximately 400 mmol) of potassium carbonate in 400 ml of tetrahydrofuran, approximately 34.4 ml of isopropylamine (400 mmol) was added. room temperature and allowed to react for 60 h and then filtered .. Potassium carbonate was washed with ethyl acetate and then the organic layer was concentrated in vacuo, which led to crystallization of the target compound, which was then isolated by filtration and washed with a small volume of hexane.

Yield: 66.37 g, yellow crystals (84%).

B. 3 -Isopropylamino-4-nitro-2 ', 3' -difluorobenzophenone.

To a cold (0 ° C) mixture of 7.65 g (50 mmol) of 2,3 difluorophenylacetonitrile and 9.9 g (50 mmol) of the compound of Example 1A in 80 ml of dimethylformamide was added 11.22 g (100 mmol) of potassium tert-butoxide. The reaction mixture was heated at room temperature and allowed to react for about 1 hour. When the reaction was substantially complete, as determined by TLC, the mixture was cooled to 0 ° C, then 15 ml of a 30% hydrogen peroxide solution was added. The mixture was warmed to room temperature, stirred overnight and then poured into 1 L of 1N. hydrochloric acid to obtain 16 g of an orange solid, which was used without further purification.

C. 3-Isopropylamino-4-amino-2 ', 3'-difluorobenzophenone.

The compound of Example 1B was hydrogenated in 250 ml of tetrahydrofuran using 2.1 g of Raney nickel catalyst at 60 ρδΐ (413685.45 Pa) hydrogen (gas) for six hours. The reaction mixture was filtered and the filtrate was concentrated in vacuo to give 14 g of a solid, which was used without further purification.

Ώ.

To a cold (0 ° C) mixture of 14 g of 1C pimer in 125 ml of isopropyl alcohol was added one equivalent of cyanogen bromide (9.6 ml of a 5M solution in acetonitrile). The resulting mixture was warmed to room temperature and stirred for 2 days and then concentrated in vacuo to give a residue. This residue was redissolved in ethyl acetate and then sonicated to obtain 13.0 g of crystals. Analysis for C ₁₇ H ₁₆ N ₃ Ovgr ₂ :

Calculated: C 51.53; H 4.07; N, 10.61; Wh 20.17;

Found: C, 51.64; H 4.17; N, 10.51; Wh 20.41.

Μδ (ΕΏ): 315 (M ⁺ ).

¹ H ΝΜΒ (300 ΜΗζ; ά ₆ -ΏΜ8Θ): δ 1.56 (ά, 6H); 4.85 (grain! E1, 1H); 7.41 (t, 2H); 7.33 (ά, 1H); 7.67 (ά, 1H); 7.74 (t, 1H); 8.01 (δ, 1H); 8.87 (δ, 2H).

1E (СНС1 ₃ ): υ 3088, 2984, 1663, 1626, 1481, 1304, 1276 st ^-1 .

IU / U18 (95% E1OH): λ ^ χ = 318 pt (E = 11480); 223 pt (E = 24524).

E p

1E (СНС1 ₃ ): ν 3380, 2910, 1652, 1608, 1522, 1307, 1276, 1264 st ^-1 .

υν / νΐδ (95% Е1ОН): λ _ΟΗΧ = 341 pt (8 = 21011); 220.5 pt (E = 26966).

Ε

To a cold (-78 ° C) solution of 18.8 ml (76 mmol) of bis (trimethylsilyl) acetamide in 200 ml of tetrahydrofuran, 30.4 ml of 2.4 M n-butyllithium in hexane (76 mmol) was slowly added, followed by 3 0 g (9.5 mmol) of Example 1E. The reaction mixture was stirred for 8 hours at -78 ° C and then allowed to warm to room temperature. After completion of the reaction, by HPLC, the reaction was quenched by the addition of 6.4 yl (76 mmol) of concentrated hydrochloric acid and then concentrated in vacuo to give an oil, which was then redissolved in formic acid containing 1% concentrated hydrochloric acid. The mixture was allowed to react for 4 hours at 95 ° C. When the reaction was substantially completed by HPLC, the mixture was concentrated in vacuo to give an oil. This oil was separated by reverse phase HPLC (RP-HPLC) (eluent acetonitrile in water) to give the cis and trans isomers of the target compound.

Cis is not characterized by trans

Analysis for C ₁₉ H ₂ ^ ₄ O ₂ E ₂ C1: Calculated: C 55.55; H 5.15; N 13.64; Found: C, 54.21; H 4.93; N, 12.98.

Μδ (ΕΏ): 356 (M ⁺ ). ¹ H NΜΒ (300 Μ &; ά6ΏΜ8Ο): δ 1.48 (ά, 6H); 4.73 (8er1e1, 1H); 6.71 (δ, 1H); 7.93 (t, 3H); 7.18 (t, 2H); 7.25 (ά, 1H); 7.35 (t, 1H); 7.42 (δ, 1H); 7.52 (δ, 1H); 7.79 (δ, 2H). ΙΕ (KVg): υ 3152, 2982, 1662, 1596, 1483, 1474, 1269 st ^-1 .

υν / νΐδ (95% E1OH): λ ^ = 310 pt (E = 9665); 223 pt (E = 24308).

Example 2

ρ

C = s

ΝΗ-CH:

This compound was prepared essentially as described in Example 1Ε using Νmethyl-Y-trimethylsilylacetamide.

Cis is not characterized by trans

Analysis for C ₂₀ H ₂₃ ^ O ₂ E ₂ C1:

The target compound was obtained by adding 1N. sodium hydroxide to the compound obtained in example 11) in ethyl acetate. The resulting layers were separated and the organic phase was concentrated in vacuo.

Yield: 9.34 g (62%)

Analysis for C ₁₇ H ₁₅ ^ OE ₂ :

Calculated: C 64.76; H 4.80; N, 13.33;

Found: C, 64.97; H 4.78; N, 13.40.

Μδ (ΕΏ): 315 (M ⁺ ). ¹ N NΜΒ (300 Μ #ζ ά ₆ ΏΜδΟ): δ 1.38 (ά, 6Н); 3.67 (grit1e1, 1H); 7.01 (δ,

2H); 7.18 (ά, 1H); 7.35 (t, 3H); 7.66 (δ, 1H); 7.77 (δ, 1H).

Calculated: C 56.54; H 5.46; N 13.19;

Found: C 56.32, H 5.10; N 13.06.

Μδ (ΕΏ): 370.3 (M ⁺ ).

¹ N ΝΜΚ (300 ΜΗζ; ά ₆ -ΏΜ8Ο): δ 2.52 (ά, 6Н); 2.59 (ά, 3H); 4.81 (grit1e1, 1H); 6.78 (δ, 1H); 6.94 (t, 2H); 7.2 (t, 1H); 7.4 (t, 2H); 7.6 (δ, 1H); 8.19 (t, 1H); 8.75 (t, 1H); 8.79 (δ, 1H).

ΙΕ (KVg): υ 3068, 1669, 1627, 1589, 1482, 1474 st ^-1 . IU / U18 (95% E1OH): / max = 305 pt (E = 13688); 223 pt (E = 30904).

Example 3

g

OS · HC1

Eun-CH ₂ C? 1 ₂

The compound was prepared essentially according to Example 1E, except that n-butyllithium (15.85 mmol) was slowly added to the solution, which was prepared as follows. A cold (-78 ° С) solution of bis (trimethylsilyl) amide (1 equivalent) and ethyl acetamide (1 equivalent) in tetrahydrofuran was stirred for 1 h, followed by the addition of chlorotrimethylsilane (1 equivalent). The resulting solution was stirred for 15 minutes and then allowed to warm slowly to room temperature. Note: the solution was cooled again to -78 ° C before adding n-butyllithium.

Cis is not characterized by trans

Analysis for C ₂₁ H ₂₂ 0OT ₂ —HC1-H ₂ O: Calculated: C 57.47; H 5.74; N, 12.76; Found: C 57.25; H 5.65; N, 12.74. Μδ (ΕΏ): 384.2 (M ⁺ ).

¹ N NΜΕ (300 Μ11ζ; ά ₆ -ΏΜ8Ο): δ 0.96 (1, 3H); 1.49 (ά, 6H); 3.0 (p, 2H); 4.80 ^ ep1e1, 1H);

6.74 (δ, 1H); 6.88 (1, 1H); 6.94 (ά, 1H); 7.16 (ς, 1H); 7.30-7.44 (t, 2H); 7.55 (δ, 1H); 8.17 (1, 1H);

8.75 (δ, 2H); 12.8 (δ, 1H).

ΙΕ (СНС1 ₃ ): υ 2986, 1664, 1602, 1514, 1482 st ^-1 .

υν / νΐδ (95% E1OH): = 304.00 pt (E = 13407.11); 224.00 pt (E = 31891.73).

The following compounds were prepared essentially as described in Example 1Α-Ε.

Example 4

O = C -CH1 νη ₂ cis

M5 (ΕΏ): 338 (M ⁺ ).

Analysis for C ₁₉ H ₁₉ 0 OT-HC1:

Calculated: C 60.88; H 5.38; N, 14.95;

Found: C, 60.62; H 5.66; N, 14.78. trance

Μ δ (Εϋ): 338 (M ⁺ ). Analysis for

C ₁₉ H ₁₉ 0 OE-HCE1,2 H ₂ O:

Calculated: C 57.56; H 5.70; N, 14.13;

Found: C, 57.26; H 5.28; N, 13.75.

cis

Analysis for C ₂₃ H ₁₈ 0 O ₂ P ₂ :

Calculated: C 65.71; H 4.32; N, 13.33;

Found: C, 65.44; H 4.30; N 13.05.

Μδ (ΕΏ): 420 (M ⁺ ).

¹ N NΜΕ (250 ΜΠζ; ά ₆ - ^ ΜδΟ): δ 3.83 (δ, 3Н); 6.08. (δ, 1H); 6.29 (δ, 2H); 6.78 (t, 2H); 7.13 (t, 6H); 7.33 (t, 4H). ΙΕ (СНС1 ₃ ): υ 3390, 3013, 1664, 1514, 1254 st ^-1 .

υν / νΐδ (95% Е1ОН): / _max = 217 pt (E = 40891).

Trance

Analysis for C ₂₃ H ₁₈ 0 O ₂ T ₂ :

Calculated: C 65.71; H 4.32; N, 13.33;

Found: C, 63.22; H 4.63; N, 12.63.

Μδ (ΕΏ): 420 (M ⁺ ).

¹ N NΜΕ (250 Μ11ζ; ά ₆ - ^ ΜδΟ): δ 3.86 (δ, 3H); 6.40 (δ, 2H); 6.49 (δ, 1H); 6.71 (ά, 1H); 6.84 (t, 3H); 7.13 (t, 4H); 7.32 (t, 3H); 7.36 (ά, 1H). ΙΕ (KBg): υ 3416, 3314, 3201, 1664, 1582, 1543, 1513, 1271 st ^-1 .

υν / νΐδ (95% Е1ОН): / max = 330 pt (E = 16300); 226 pt (E = 33818).

Example 6

cis is not characterized by trans

Μδ (ΕΏ): 382 (M ⁺ ).

Analysis for C ₂₁ H ₂₀ 0OE ₂ —NSE1,2H ₂ O:

Calculated: C 57.26; H 5; N, 12.72;

Found: C 57.21; H 5.08; N, 12.47.

Example 7

I - HC1 νη ₂ cis

Μδ (ΕΏ): 364 (Μ ⁺ ).

Analysis for C ₂₁ H ₂₁ 0OT-1.2HC1: Calculated: C 61.79; H 5.48; N 13.73;

Found: C, 61.68; H 5.60; N, 13.56. trance

Μδ (ΕΏ): 364 (M ⁺ ).

Analysis for C ₂ | 11 ₂ | Ν | Ο1 ^; Ί 1C1:

Calculated: C 62.92; H 5.53; N 13.98;

Found: C, 62.78; H 5.55; N, 13.68.

The following compounds were prepared essentially as described above in Example 2.

Example 8

cis

Μ8 (ΓΌ): 378 (M ⁺ ).

Analysis for C ₂₂ H ₂₃ ^ OH-HC1-0.5H ₂ O: Calculated: C 62.33; H 5.94; N 13.22;

Found: C, 62.33; H 5.74; N, 12.98.

trance

Μ8 (ΓΌ): 378 (M ⁺ ).

Analysis for C ₂₂ H ₂₃ ^ OR-HC1-0.2H ₂ O: Calculated: C 63.14; H 5.88; N 13.39;

Found: C, 63.00; H 5.92; N, 13.33.

Example 9

Eun-SNs cis

Μ8 (ΓΌ): 350 (M ⁺ ).

Analysis for C ₂₀ H ₁₉ ^ OR-1.5CH1-1.5H ₂ O: Calculated: C 55.59; H 5.48; N, 12.97;

Found: C 55.92; H 5.24; N, 12.80.

trance

Μ8 (ΓΌ) 350 (Μ ⁺ ).

Analysis for C ^ Hf ^ OR-DBNSy Calculated: C 58.77, H 5.08; N 13.71; Found: C, 58.89; H 5.42; N, 12.55. Example 10

cis is not characterized by trans

Μ8 (ΓΌ): 366 (M ⁺ ).

Analysis for C ₂₁ H ₂₃ ^ OG-1.4NS1:

Calculated: C 60.42; H 5.89; N 13.42;

Found: C, 60.28; H 6.15; N, 13.24.

The following compounds were prepared essentially as described in detail in Example 1AP.

Example 11

These compounds inhibit viral RNA plus chain replication by interfering with the structure and / or function of the virus replication complex (membrane-bound complex of viral and cellular proteins). Mutant rhinovirus and enterovirus have been isolated that exhibit very low levels of drug resistance. These mutants contain a single amino acid substitution in a protein that is expressed by a viral gene known as ZA. Thus, the compounds of this invention inhibit rhinovirus and enterovirus by inhibiting the function of 3A. Gene 3A encodes a hydrophobic protein that serves as a scaffold protein that attaches the proteins of the replication complex to intracellular membranes.

The replication strategy for flaviviruses such as hepatitis C virus (HCV) and bovine diarrhea virus (VUEU) is similar to the strategy of rhinovirus and enterovirus discussed above. In particular, both families of viruses contain single-stranded informational sense RNA, which is replicated to the cytoplasmic complex through an intermediate RNA minus chain. In addition, both families of viruses translate their genome into a polyprotein, which is then cleaved. In addition, the replication complexes of both viruses are tightly bound to intracellular membranes. Finally, both virus families have similar genomic structures, including the presence of the 5'- and 3'-untranslated region, which viruses require for replication. There are two NSU proteins that are involved in this intracellular association: N82 and N84. It is postulated that either N82 or N84 is similar to picornavirus 3A protein.

Accordingly, another embodiment of the present invention is a method of treating or preventing a flavivirus infection, comprising administering to a host in need of treatment an effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof. This compound preferably inhibits hepatitis C.

As noted above, the compounds of this invention are useful as antiviral agents. They found inhibitory activity against various enteroviruses and rhinoviruses. One embodiment of the invention is a method for treating or preventing a picornavirus infection, comprising administering to a host in need of treatment 19 an effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof.

The term effective amount, as used herein, means an amount of a compound of formula I that is capable of inhibiting viral replication. The inhibition of picornavirus contemplated by this method involves either therapeutic or prophylactic treatment, as the case may be. The specific dose of an administered compound according to this invention to obtain therapeutic or prophylactic effects will, of course, be determined by the specific circumstances of the particular case, including, for example, the compound administered, the route of administration, the condition to be treated, and the individual to be treated. A typical daily dose is from about 0.01 mg / kg to about 50 mg / kg (active compound of the present invention per kg of body weight). Preferred daily doses will typically be in the range of about 0.05 mg / kg to about 20 mg / kg, and ideally, about 1 mg / kg to about 10 mg / kg.

These compounds can be administered in various ways, including, orally, rectally, transdermally, subcutaneously, intravenously, intramuscularly and intranasally (through the nose). The compounds of this invention are preferably prepared before administration. Thus, another object of the present invention is a pharmaceutical composition comprising an effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, diluent or excipient.

The active ingredient in such preparations is from 0.1% to 99.9% by weight of the drug. By pharmaceutically acceptable is meant that the carrier, diluent or excipient are compatible with the other ingredients of the drug and are not harmful to the person taking the drug (recipient).

These pharmaceutical preparations are prepared by known procedures using well known and readily available ingredients. In preparing the compositions of this invention, the active ingredient is usually mixed with a carrier or diluted with a carrier or placed inside a carrier, which may be in the form of a capsule, pillow, paper or other container. When this carrier serves as a diluent, it may be a solid, semi-solid or liquid material that acts as a carrier, excipient or medium for the active ingredient. So, these compositions can be in the form of tablets, pills, powders, lozenges, pads, starch wafers, elixirs, suspensions, emulsions, solutions, syrups, aerosols (in solid form or in liquid medium), ointments containing, for example, up to 10% by weight of the active compound, soft and hard gelatin capsules, suppositories, sterile injectable solutions, sterile packaged powders, etc.

The following preparation examples are illustrative only and do not limit the scope of the invention. The term active ingredient refers to a compound of formula I or a pharmaceutically acceptable salt thereof.

Composition 1.

Hard gelatin capsules are prepared using the following ingredients:

Amount (mg / capsule)

Active Ingredient250

Dried starch200

Magnesium Stearate10

Total 460 mg

Composition 2.

A tablet is prepared using the ingredients listed below:

Amount (mg / capsule)

Active Ingredient250

Microcrystalline Cellulose 400

Colloidal silicon dioxide 10

Stearic acid5

Total 665 mg

These components were mixed and pressed to form tablets with a weight of each tablet of 665 mg.

Composition 3.

An aerosol solution containing

following components: Weight Active ingredient 0.25 Methanol 25.75 Propellant 22 (Chlorodifluoromethane) 70.00 Total 100.00

The active compound is mixed with ethanol and the mixture is added to a portion of the propellant 22, cooled to -30 ° C, and transferred to a filling device. Then the required amount is fed into a stainless steel container and diluted with the remaining part of the propellant. Then, a valve element is mounted on this container.

Composition 4.

Tablets, each containing 60 mg of the active ingredient, are prepared as follows:

Amount (mg / tablet)

Active ingredient 60

Starch 45

Microcrystalline Cellulose 35

Polyvinylpyrrolidone (as a 10% solution in water) 4

Sodium Carboxymethyl Starch 4.5

Magnesium Stearate 0.5

Talc 1

Total150

The active ingredient, starch and cellulose are passed through a Νο sieve. 45 tech 11 and 8. and thoroughly mixed. An aqueous solution containing polyvinylpyrrolidone is mixed with the obtained powder, and then the mixture is passed through a Νο sieve. 14 stream and. 8. The granules thus obtained are dried at 50 ° C. and passed through a Νο sieve. 18 stream and. 8. Then sodium carboxymethyl starch, magnesium stearate and talc, previously passed through a Νο sieve. 60 tech 11 and 8., are added to these granules, which, after mixing, are pressed on a tablet machine to produce tablets, each of which has a weight of 150 mg.

Composition 5.

Capsules, each of which contains 80 mg of the active ingredient, are prepared as follows:

Amount (mg / capsule) Active ingredient 80 mg

Starch 59 mg

Microcrystalline Cellulose 59 mg

Magnesium Stearate 2 mg

200 mg total

The active ingredient, cellulose, starch and magnesium stearate are mixed, passed through a Νο sieve. 45 tech 11 and 8. and 200 mg hard gelatin capsules are filled with this mixture in each capsule.

Composition 6.

Suppositories, each of which contains 225 mg of active ingredient, are prepared as follows:

Active ingredient 225 mg

G saturated fatty acid glycerides 2000 mg Total 2225 mg

The active ingredient is passed through a Νο sieve, 60 tek11 and 8. and suspended in saturated fatty acid glycerides, previously melted using minimal heat, if necessary. Then this mixture is poured into a suppository mold with a nominal capacity of 2 g and allowed to cool.

Composition 7.

Suspensions, each of which contains 50 mg of the active ingredient per dose of 5 ml, are prepared as follows:

Active ingredient 50 mg

Sodium Carboxymethyl Cellulose 50 mg

Syrup 1.25 ml

Benzoic acid solution 0.10 ml

Fragrance s. | .U.

(as much as you like) Dye s. | .u.

Purified water to a final volume of 5 ml

The active ingredient is passed through a sieve Νο 45 tech11 and 8. and mixed with sodium carboxymethyl cellulose and syrup to form a smooth paste. The benzoic acid solution, flavor (flavor and odor improving substance) and color are diluted with a portion of water and added with stirring. Then add a sufficient amount of water to obtain the desired volume.

Composition 8.

An intravenous composition can be prepared as follows:

Active ingredient 100 mg

Isotonic saline 1000 ml

A solution of the above ingredients is usually administered intravenously to a subject at a rate of 1 ml per minute.

The following experiment is conducted to demonstrate the ability of compounds of formula I to inhibit a specific virus.

Test method for analysis of action against picornavirus

African green monkey kidney cells (B8C-1) or Heb cells (5-3) were grown in 25 ml Falcon flasks at 37 ° C in 199 medium with 5% inactivated fetal calf serum (EB8), penicillin (150 units per ml) and streptomycin (150 micrograms per milliliter (μg / ml). When confluent monolayers formed, the growth medium was removed as supernatant and 0.3 ml of a suitable virus dilution was added to each flask (ECHO-, Mei ^ -, Koksaki-, polio- or rhinovirus.) After absorption for 1 h at room temperature, virus-infected glue the full-face layer was covered with a medium containing one part of 1% yiadag .ο. 2 and one part of double-strength medium 199 with EB8, penicillin and streptomycin containing the drug at concentrations of 100, 50, 25, 12, 5, 3, and 0 μg / ml. The drug-free flask served as a control for this test. Stock solutions of vinylacetylenbenzimidazole compounds were diluted to a concentration of 10 ⁴ μg / ml, then the flasks were incubated for 72 hours at 37 ° C for full, Coxsackie, ECHO and Mepdo virus and for 120 hours for rhinovirus. Viral plaques (sterile spots) were observed in those areas of the cells that were infected with the virus and where the virus was reproduced in the cells. A solution of 10% formalin and 2% sodium acetate was added to each flask to inactivate the virus and fix the cell layer on the surface of the flask. Viral plaques, regardless of size, were considered crystalline violet after staining the surrounding cell zones. The number of plaques was compared with the number in the control at each drug concentration. The activity of the test compound was expressed as a percentage reduction in the number of plaques, or percent inhibition. Alternatively, the concentration of the drug, which inhibits plaque formation by 50%, can be used as a measure of activity. 50% inhibition is illustrated by the symbol 1C ₅₀ .

Ιη νΐΐΓθ CPE / XTT analysis of action against WUBU

ΜΌΒΚ cells were distributed in a 96-well microtiter plate at 10,000 cells per well with Minimal Essential Medium containing Eagle's balanced salt solution (ΕΒ88), 2% horse serum, penicillin (100 units / ml) and streptomycin (100 μg / ml). The tablets were grown at 37 ° C in a CO ₂ thermostat overnight. Then, the ΜΌΒΚ cells infected ~ 0.02 n1 (multiplicity of infection) of the bovine viral diarrhea virus (VUOU, ATCC UK-534). After the adsorption of the virus by the cells for 1-2 hours, medium containing serial dilutions of the drug, or only medium, was added to the wells. After additional incubation for 3-4 days (when a strong cytopathogenic effect (CPE) was detected in the wells containing only the medium), the antiviral effect of the test drugs was evaluated by an XTT assay described below.

XTT [2,3-bis (methoxy-4-nitro-5-sulfophenyl) -2H-tetrazolium-5-carboxanilide, internal salt, sodium salt] at 1 mg / ml for a warm medium without BV8 was prepared and immediately used in freshly prepared form. For every 5 ml of XTT solution, 25 μl of 5 mM PMS (phenazine methosulfate) in phosphate-buffered saline was added. Then, 50 μl of a freshly prepared XTT / PMS mixture was added to each well of a microtiter plate. Incubated at 37 ° C (CO ₂ ) for 3-4 hours, or until a discoloration is evident. Absorption at 450 nm / sr. 650 nm was recorded in a spectrophotometer. Then, the concentration of the drug required for a 50% cytotoxic effect, compared with the control without the drug and without the virus (TC50), and the inhibitory development of the viral cytopathogenic effect (CPE), was determined from the linear portion of each dose-response curve (1C50).

Claims (7)

1. The compound of formula I: p θ ГР 1 ^к о = с νκ ⁴ η ⁵ where а is 0, 1, 2 or 3; each K independently represents hydrogen, halogen, cyano, amino, halogen (C ₁ -C ₆ ) alkyl, di (C ₁ -C ₄ ) alkylamino, azido, C ₁ -C ₆ alkyl, carbamoyl, carbamoyloxy, carbamoylamino, C1-C4 alkoxy , _C1-C4 alkylthio, _C1-C4 -alkilsulfinil, C1 -C4 alkylsulfonyl, pyrrolidino, piperidino or morpholino; K ⁰ is hydrogen, halogen, C ₁ -C ₄ alkyl or C ₁ -C ₄ alkoxy; K ¹ is halogen, cyano, hydroxy, methyl, ethyl, methoxy, ethoxy, methylthio, methylsulfinyl or methylsulfonyl; K ² is hydrogen, amino or —IIS (O) (C ₁ -C ₆ alkyl); K ³ is dimethylamino, C1-C10-alkyl, Cs-C ₇ cycloalkyl, substituted C ₃ -C ₇ cycloalkyl, halo (C ₁ -C ₆₎ alkyl, phenyl, substituted phenyl, furyl, thienyl, thiazolyl, thiazolidinyl, pyrrolidino , piperidino, morpholino or a group of the formula K ⁴ and K ⁵ independently represent hydrogen or _C1-C4 alkyl; or a pharmaceutically acceptable salt thereof. 2. The compound according to claim 1, wherein a is 0, 1 or 2; each K is independently hydrogen, halogen, _C1-C4 alkyl, _C1-C4-alkoxy or di (C ₁ -C 4) alkylamino; K ⁰ is hydrogen; K ² is amino; K ³ is dimethylamino, _C1-C6 alkyl, halo (C ₁ -C ₆₎ alkyl, phenyl, substituted phenyl, C ₃ -C ₇ -cycloalkyl, substituted C ₃ -C ₇ cycloalkyl, thienyl, thiazolidinyl, pyrrolidino, piperidino or morpholino; K ⁴ is hydrogen, methyl or ethyl; K ⁵ is hydrogen, methyl or ethyl; or a pharmaceutically acceptable salt thereof. 3. The compound according to claim 2, where a is 0 or 1; each K is independently hydrogen, fluoro, methyl, ethyl, methoxy, ethoxy, dimethylamino; K ³ is C ₁ -C ₄ alkyl, phenyl, substituted phenyl, C ₃ -C ₇ cycloalkyl or substituted C ₃ -C ₇ cycloalkyl; or a pharmaceutically acceptable salt thereof. 4. The compound according to claim 3, which is or a pharmaceutically acceptable salt thereof. 5. A pharmaceutical composition comprising an effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof, according to any one of claims 1 to 4, with one or more pharmaceutically acceptable carriers, diluents or excipients. 6. The use of a compound of formula I or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4 as a pharmaceutical agent. 7. The use according to claim 6, characterized in that the pharmaceutical agent is an antiviral agent.