WO2016130043A1

WO2016130043A1 - Benzo[1,2,4]thiadiazine inhibitors of hepatitis b virus replication and pharmaceutical composition for treating hepatitis b

Info

Publication number: WO2016130043A1
Application number: PCT/RU2015/000856
Authority: WO
Inventors: Александ Васильевич ИВАЩЕНКО; Андрей Александрович ИВАЩЕНКО; Николай Филиппович САВЧУК; Вадим Васильевич БЫЧКО; Александр Викторович ХВАТ; Борис РОГОВОЙ
Original assignee: Александ Васильевич ИВАЩЕНКО; Алла, Хем, Ллс; Андрей Александрович ИВАЩЕНКО; Николай Филиппович САВЧУК
Priority date: 2015-02-13
Filing date: 2015-12-08
Publication date: 2016-08-18

Abstract

The subject matter of this invention is novel inhibitors of hepatitis B virus replication, which are 1,1-dioxo-1,4-dihydro-2H-benzo[1,2,4]thiadiazine-3-ones of general formula 1, pharmaceutically acceptable salts and/or hydrates thereof. (1), where Ar¹ and Ar² are optionally identical aryls, including phenyl, which is optionally substituted with one, two or three substituents selected from C₁-C₄ alkyl, methoxyl, halogen, carboxyl of optionally substituted carbamoyl and carbonitrile.

Description

Benzo [1,2,4] thiadiazine hepatitis B virus replication inhibitors and pharmaceutical composition for the treatment of hepatitis B.

Technical field

This invention relates to new hepatitis B virus replication inhibitors (HBV), a new pharmaceutical composition for the treatment of hepatitis B, a method for their preparation and use.

State of the art

Hepatitis B is a liver infection that is potentially life threatening due to HBV. He presents one of the main problems of the global

health care. Hepatitis B can lead to the development of chronic liver disease and create a high risk of death from cirrhosis and liver cancer.

According to WHO, the highest levels of hepatitis B prevalence are found in sub-Saharan Africa and East Asia. Most people in these regions acquire hepatitis B infection in childhood, and between 5% and 10% of adults have a chronic infection.

High rates of chronic infections are also observed in the Amazon region and in southern parts of Eastern and Central Europe. It is estimated that between 2% and 5% of the total population of the Middle East and the Indian subcontinent are chronically infected. AT

In Western Europe and North America, less than 1% of the population is chronically infected.

The likelihood that a hepatitis B virus infection will become chronic depends on the age at which the person acquires the infection. Chronic infections develop in 80-90% of children infected during the first year of life and in 30-50% of children infected before the age of six. Among adults, chronic infections develop in <5% of infected, otherwise healthy people. 5–25% of adults who become chronically infected in childhood die from hepatitis B-related cancer or cirrhosis.

More than 240 million people have chronic (long-term) liver infections. About 780,000 people die each year from the acute or chronic effects of hepatitis B. A hepatitis B vaccine has been available since 1982. This vaccine is 95% effective in preventing infection and its chronic effects and is the first vaccine against one of the main types of human cancer.

People with chronic hepatitis B who need treatment are treated with antiviral drugs such as tenofovir and entecavir, as well as interferon injections. Treatment can slow the progression of cirrhosis, reduce the incidence of hepatocellular carcinoma, and improve long-term survival.

In recent years, non-nucleoside heterocyclic compounds have been discovered that inhibit the replication of hepatitis B virus [US 6436943, US 2013/0071354, WO 2013/102655], including: methyl (R) -4- (2-chloro-4-fluorophenyl) - 2- (3,5-difluoro-2-pyridinyl) - 6-methyl-l, 4-dihydro-pyrimidine-5-carboxylate (BAY 41-4109) [O. Weber et al. Inhibition of human hepatitis B virus (HBV) by a novel non-nucleosidic compound in a transgenic mouse model. Antiviral Res. 54, 69-78, 2002.], as well as sulfamoyl arylamides [WO 20013/006394, WO 2013/096744, WO 2014/033 170, WO 2014/033176], sulfamoylthiophenamides [WO 2014/184365].

Derivatives of 1, 1-dioxo-2H-1, 2,4-benzothiadiazin-3 (4H) -one are known, which have shown high activity as antagonists of the orexin-2 receptor and are claimed for the treatment and prevention of drug dependence, motor disorders, sleep disorders, neurological diseases associated with depression, cognitive disorders, Alzheimer's disease. A method for their preparation by the interaction of 2-nitrobenzene sulfonyl chloride and substituted aniline is also described, followed by reduction of the nitro group in the presence of tin dichloride and cyclization in the presence of triphosgene [WO 2014006402]. The solid-phase synthesis of 2,4-disubstituted 1, 2,4-benzothiadiazin-3-one 1, 1-dioxides from 4-nitrophenylacetic acid is known [Shingo Makino et al. Efficient synthesis of 2,4-disubstituted 1, 2,4-benzothiadiazin-3-one 1, 1 -dioxides on solid support. J. Comb. Chem. 5 (1), 73-78, 2003].

However, there is no specific treatment for acute hepatitis B to date. In this regard, the search for new drugs for the treatment of hepatitis B is an urgent task. Disclosure of invention

The following are definitions of terms that are used in the description of this invention.

“Alkyl” means an aliphatic hydrocarbon linear or branched group with 1 to 12 carbon atoms in the chain. Branched means that the alkyl chain has one or more "lower alkyl" substituents. Alkyl may have one or more, same or different substituents, including halo, alkenyloxy, cycloalkyl, aryl, heteroaryl, heterocyclyl, aroyl, cyano, hydroxy, alkoxy, carboxy, alkynyloxy, aralkoxy, aryloxy, aryloxycarbnyl, alkylthio, heteroarylthio, aralkyl alkylsulfonylheteroaralkyloxy, annelated heteroarylcycloalkenyl, annelated heteroarylcycloalkyl, annelated heteroarylheterocyclenyl, annelated heteroarylheterocyclyl, annelated arylcycloalkenyl, annelated a rilcycloalkyl, annelated arylheterocyclenyl, annelated arylheterocyclyl, alkoxycarbonyl, aralkoxycarbonyl, heteroaralkyloxycarbonyl or

R _k ^a R _{k +} i ^a NS0 ₂ -, where R _k ^a and R _{k +} i ^{a are} independently “amino substituents”, for example, a hydrogen atom, alkyl, aryl, aralkyl, heteroaralkyl, heterocyclyl or heteroaryl, or Rk ^a and RH-I ³ together with the N atom to which they are bonded form through R _k ^a and R _{k +} i ^a 4 - 7 membered heterocyclyl or heterocyclenyl. Preferred alkyl groups are methyl, trifluoromethyl, cyclopropylmethyl, cyclopentylmethyl, ethyl, n-propyl, iso-propyl, n-butyl, tert-butyl, n-pentyl, 3-pentyl, methoxyethyl, carboxymethyl, methoxycarbonylmethyl, ethoxycarbonylmethyl, benzyloxycarbonylcarbonyl and pyridylmethyloxycarbonylmethyl. The preferred "alkyl substituents" are cycloalkyl, aryl, heteroaryl, heterocyclyl, hydroxy, alkoxy, alkoxycarbonyl, aralkoxy, aryloxy, alkylthio, heteroarylthio, aralkylthio, alkylsulfonyl, arylsulfonyl, alkoxycarbonyl, aralkoxycarbonyl, geteroaralkiloksikarbonil or ^{_{^{Rk a R k + i a N-}}} , Rk ^a Rk ₊ i ^a NC (= 0) -, annelated arylheterocyclenyl, annelated arylheterocyclyl. “Alkoxy” or “alkyloxy” means an alkyl-O— group in which alkyl is defined in this section. Preferred alkyloxy groups are methoxy, ethoxy, n-propoxy, iso-propoxy and n-butoxy.

“Aryl” means an aromatic monocyclic or polycyclic system comprising from 6 to 14 carbon atoms, preferably from 6 to 10 carbon atoms. Aryl may contain one or more “cyclic system substituents”, which may be the same or different. Representative aryl groups are phenyl or naphthyl, substituted phenyl or substituted naphthyl. Aryl can be annelated with a non-aromatic ring system or heterocycle.

“Halogen” means fluoro, chloro, bromo and iodo. Fluorine, chlorine and iodine are preferred.

“Heterocyclyl” means an aromatic or non-aromatic saturated monocyclic or polycyclic system comprising from 3 to 10 carbon atoms, preferably from 5 to 6 carbon atoms, in which one or more carbon atoms are replaced by a heteroatom such as nitrogen, oxygen, sulfur. The prefix "aza", "oxa" or "thia" before heterocyclyl means the presence in the cyclic system of a nitrogen atom, an oxygen atom or a sulfur atom, respectively. Heterocyclyl may have one or more “cyclic system substituents,” which may be the same or different. The nitrogen and sulfur atoms in the heterocyclyl can be oxidized to Ν-oxide, S-oxide or S-dioxide. Representatives of heterocyclyl are piperidine, pyrrolidine, piperazine, morpholine, thiomorpholine, thiazolidine, 1, 4-dioxane, tetrahydrofuran, tetrahydrothiophene, etc.

"Hydrate" means a stoichiometric or non-stoichiometric composition of a compound or its salt with water.

“Urea carbamoyl” means a substituent attached to a carbamoyl group, the meaning of which is defined in this section. The carbamoyl substituent is hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, alkoxycarbonylalkyl, aralkoxycarbonylalkyl, heteroaralkyloxycarbonylalkyl or R _k ^a R _{k +} i ^a N-,

annelated heteroarylcycloalkenyl, annelated heteroarylcycloalkyl, annelated heteroarylheterocyclenyl, annelated heteroarylheterocyclyl, annelated arylcycloalkenyl, annelated arylcycloalkyl, annelated arylheterocyclenyl, annelated arylheterocyclyl. Preferable "carbamoyl substituents" are alkyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, alkoxycarbonylalkyl, aralkoksikarbonilalkil, geteroaralkiloksikarbonilalkil or _{^{_{^{R k a R k + i a}}}} N-, Jak ^and k + 1 ~ C (= 0) -alkyl, annelated arylheterocyclenyl, annelated aryl heterocyclyl.

“Substituent cyclic system” means a substituent attached to an aromatic or non-aromatic cyclic system, including hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, heteroaralkyl, hydroxy, hydroxyalkyl, amino, aminoalkyl, alkoxy, aryloxy, acyl, aroyl, halogen , nitro, cyano, carboxy, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, alkyloxyalkyl, aryloxyalkyl, heterocyclyloxyalkyl, arylalkyloxyalkyl, heterocyclylalkyl-hydroxyalkyl, alkylsulfonyl, arylsulfonyl, heterocylalkyl lfinil, arylsulfinyl, geterotsiklilsulfinil, alkylthio, arylthio, heterocyclylthio, alkylsulfonylalkyl, arylsulfonylalkyl, geterotsiklilsulfonilalkil, alkilsul- finilalkil, arylsulfinylalkyl, geterotsiklilsulfinilalkil, alkylthioalkyl, arylthioalkyl geterotsikliltioalkil, arilalkilsulfonilalkil, geterotsiklilalkil- sulfonylalkyl, arilalkiltioalkil, geterotsiklilalkiltioalkil, cycloalkyl, cycloalkenyl, heterocyclyl, heterocyclenyl, amidino, R _k ^a R _{k +} i ^a N-, R _k ^a N =, R ^ _; ^a R _{k + l} ^a N-alkyl-,

or R _k ^a R _{k +} i ^a NS0 ₂ -, where R _k ^a and R _{k +} i ^a are independently amino substituents, for example, hydrogen, optionally substituted alkyl, optionally substituted aryl, optionally substituted aralkyl, or optionally substituted heteroaralkyl, or a substituent R _k ^a R _k + i ^a N-, in which R _k ^a may be acyl or aroyl, and the value R _{k +} i ^{a is as} defined above, or “cyclic system substituents” are

or R _k ^a R _{k +} i ^a NS0 ₂ -, in which R _k ^a and R _{k +} i ^a together with the nitrogen atom to which they are bonded form through R _k ^a and R _{k +} i ^a 4-7 membered heterocyclyl or heterocyclenyl.

“Carbamoyl” means C (= 0) NR _k ^a R _{k +} i ^a is a group. Carbamoyl may have one or more of the same or different "carbamoyl substituents" R _k ^a and R _k + A including hydrogen, alkenyl, alkyl, aryl, heteroaryl, heterocyclyl.

“Carboxyl” means HOC (= 0) - (carboxyl) group. “Medicinal substance” (drug substance, drug substance) means a physiologically active substance of synthetic or other (biotechnological, plant, animal, microbial and other) origin, having pharmacological activity and is the active principle of a pharmaceutical composition used for the manufacture and manufacture of a drug drug (funds).

“Medicinal product (preparation)” - a substance (or a mixture of substances in the form of a pharmaceutical composition) in the form of tablets, capsules of injections, ointments and other finished forms, intended to restore, correct or alter the physiological functions in humans and animals, as well as for the treatment and disease prevention, diagnosis, anesthesia, contraception, cosmetology and more.

"Lower alkyl" means a linear or branched alkyl with 1 to 4 carbon atoms.

A “therapeutic cocktail” is a simultaneously administered combination of two or more drugs with a different mechanism of pharmacological action and aimed at different biological targets involved in the pathogenesis of the disease.

"Pharmaceutical composition" means a composition comprising a compound of formula 1 and at least one of the components selected from the group consisting of pharmaceutically acceptable and pharmacologically compatible excipients, solvents, diluents, carriers, excipients, distributing and perceiving agents, agents delivery, such as preservatives, stabilizers, fillers, grinders, moisturizers, emulsifiers, suspending agents, thickeners, sweeteners, perfumes, flavors, antibacterial agents, fungicides, lubricants, prolonged delivery regulators, the choice and ratio of which depends on the nature and method of administration and dosage. Examples of suspending agents are ethoxylated isostearyl alcohol, polyoxyethylene, sorbitol and sorbitol ether, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, as well as mixtures of these substances. Protection against the action of microorganisms can be achieved using a variety of antibacterial and antifungal agents, for example, such as parabens, chlorobutanol, sorbic acid and the like. The composition may also include isotonic agents, for example, sugars, sodium chloride and the like. The prolonged action of the composition can be achieved using agents that slow down the absorption of the active principle, for example, aluminum monostearate and gelatin. Examples of suitable carriers, solvents, diluents and delivery vehicles are water, ethanol, polyalcohols, and also mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters (such as ethyl oleate). Examples of excipients are lactose, milk sugar, sodium citrate, calcium carbonate, calcium phosphate and the like. Examples of grinders and distributors are starch, alginic acid and its salts, silicates. Examples of lubricants are magnesium stearate, sodium lauryl sulfate, talc, and high molecular weight polyethylene glycol. The pharmaceutical composition for oral, sublingual, transdermal, intramuscular, intravenous, subcutaneous, local or rectal administration of the active principle, alone or in combination with another active principle, can be administered to animals and humans in a standard administration form in the form of a mixture with traditional pharmaceutical carriers. Suitable unit dosage forms include oral forms such as tablets, gelatine capsules, pills, powders, granules, chewing gums and oral solutions or suspensions, sublingual and buccal administration forms, aerosols, implants, local, transdermal, subcutaneous, intramuscular, intravenous, intranasal or intraocular administration forms and rectal administration forms.

“Pharmaceutically acceptable salt” means the relatively non-toxic organic and inorganic salts of the acids and bases of the present invention. These salts can be obtained in situ during the synthesis, isolation or purification of the compounds or prepared specially. In particular, base salts can be prepared on the basis of the purified free base of the claimed compound and a suitable organic or inorganic acid. Examples of salts thus obtained are hydrochlorides, hydrobromides, sulfates, bisulfates, phosphates, nitrates, acetates, oxalates, valeriates, oleates, palmitates, stearates, laurates, borates, benzoates, lactates, tosylates, citrates, maleates, fumarates, succinates, tartrates, mesylates, malonates, salicylates, propionates, ethanesulfonates, benzenesulfonates, sulfamates and the like (Detailed description of the properties of such salts is given in Berge SM, et al., "Pharmaceutical Salts" J. Pharm. Sci. 1977, 66: 1 - 19). Salts of the claimed acids can also be specially prepared by reacting the purified acid with a suitable base, and metal and amine salts can be synthesized. Metal salts include sodium, potassium, calcium, barium, zinc, magnesium, lithium and aluminum salts, the most desirable of which are sodium and potassium salts. Suitable inorganic bases from which metal salts can be obtained are hydroxide, carbonate, sodium bicarbonate and hydride, potassium hydroxide and bicarbonate, potash, lithium hydroxide, calcium hydroxide, magnesium hydroxide, zinc hydroxide. As organic bases from which salts of the claimed acids can be obtained, amines and amino acids are selected that are sufficiently basic to form a stable salt and are suitable for medical use (in particular, they should have low toxicity). Such amines include ammonia, methylamine, dimethylamine, trimethylamine, ethylamine, diethylamine, triethylamine, benzylamine, dibenzylamine, dicyclohexylamine, piperazine, ethylpiperidine, tris (hydroxymethyl) aminomethane and the like. In addition, tetraalkylammonium hydroxides, for example, such as choline, tetramethylammonium, tetraethylammonium and the like, can be used for salt formation. As amino acids, the main amino acids can be used - lysine, ornithine and arginine.

The subject of this invention is new inhibitors of hepatitis B virus replication, which are 1, 1-dioxo-1, 4-dihydro-2H-benzo [1, 2,4] thiadiazin-3-ones of general formula 1, their pharmaceutically acceptable salts and / or hydrates.

one where Ar ¹ and Ar ² are optionally the same aryl, including phenyl, optionally substituted with one, two or three substituents selected from C | - C ₄ alkyl, methoxy, halogen, carboxyl optionally substituted carbamoyl and carbonitrile.

The compounds of general formula 1, as the authors unexpectedly found, were effective inhibitors of hepatitis B virus replication.

According to the present invention, preferred inhibitors of the general formula 1 are 1, 1-dioxo-1, 4-dihydro-2H-benzo [1, 2,4] thiadiazin-3-ones 1.1-1.15

1.11 1.12 1.13 1.14 1.15

Hepatitis B virus replication inhibitors of general formula 1 are commercially available compounds from a number of companies, including ChemDiv, Inc.

(USA) [SciFinder® database from Chemical Abstracts Service].

Inhibitory activity of compounds of the general formula 1 (inhibition of secretion

The "e" antigen (HBeAg) in a culture of HBV-infected HepG2 NTCP cells) was determined by ELISA according to the protocol described below in Example 1, and their cytotoxicity was determined by the protocol described below in Example 2.

According to the present invention, new inhibitors of the general formula 1 are a drug for preparing a pharmaceutical composition and formulations for the prevention and treatment of HBV in warm-blooded animals and humans.

The subject of this invention is a pharmaceutical composition in the form of tablets, capsules or injections in a pharmaceutically acceptable package for the treatment of hepatitis B in humans and warm-blooded animals, comprising a therapeutically effective amount of an HBV replication inhibitor of general formula 1.

Pharmaceutical compositions may include pharmaceutically acceptable excipients. By pharmaceutically acceptable excipients are meant diluents, excipients and / or carriers used in the pharmaceutical field. The pharmaceutical composition along with the inhibitor of the general formula 1 or their pharmaceutically acceptable salt and / or hydrate of the present invention may include other active substances, provided that they do not cause undesirable effects. If necessary, the use of the pharmaceutical composition of the present invention in clinical practice, it can be mixed with traditional pharmaceutical carriers.

The carriers used in the pharmaceutical compositions of the present invention are carriers that are used in the pharmaceutical industry to obtain common forms, including: in oral forms, binders, lubricants, disintegrants, solvents, diluents, stabilizers, suspending agents, colorless are used agents, flavoring agents of taste; antiseptic agents, solubilizers, stabilizers are used in injection forms; in local forms, bases, diluents, lubricants, antiseptic agents are used.

The subject of this invention is a method for preparing a pharmaceutical composition by mixing with an inert excipient and / or solvent of at least one inhibitor of general formula 1 or a pharmaceutically acceptable salt thereof in a therapeutically effective amount.

The subject of the present invention is also therapeutic cocktails for treating influenza, comprising, as one of the components, a new drug or a new pharmaceutical composition containing, as an active component, at least one compound of general formula 1 or a pharmaceutically acceptable salt and / or hydrate thereof.

A therapeutic cocktail for treating HBV, along with the pharmaceutical composition of this invention, may include other known drugs for treating hepatitis B virus, or drugs that enhance the patient’s immune system.

In accordance with this invention, a method for the prevention and treatment of hepatitis B in animals and humans consists in introducing to the patient a new pharmaceutical composition or a new therapeutic cocktail.

Medicines may be administered via an inhaler, orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally or topically). The clinical dosage of an agent of general formula 1 in patients can be adjusted depending on the therapeutic efficacy and bioavailability of the active ingredients in the body, their metabolic rate and excretion from the body, as well as depending on the age, sex and stage of the patient’s disease, while the daily dose in adults is usually 10 ~ 500 mg. Therefore, when preparing the inhibitor of the present invention in the form of dosage units from the pharmaceutical composition, the above effective dosage must be taken into account, with each dosage unit containing 10-500 mg of the inhibitor of general formula 1. In accordance with the instructions of a doctor or pharmacist, these drugs may be taken times during certain periods of time.

The subject of this invention is also a method of inhibiting the activity of HBV in vivo, comprising the step of contacting an inhibitor of general formula 1 and HBV. The invention is illustrated, but not limited to the following examples.

Example 1. Determination of the antiviral activity of a pharmaceutical composition containing one of the inhibitors of general formula 1 as an active substance against hepatitis B viruses (HBV) in a human hepatoma cell culture (HepG2 NTCP) infected with HBV in vitro (Elife 2012 Nov 13; 1 : e00049. doi:

10.7554 / eLife.00049).

To determine the activity of the inhibitors, an in vitro immune ELISA test for HBV "e" antigen (HBeAg) was used. Using this test was measured

the effectiveness of inhibition by test compounds of antigen production (and therefore virus replication) in cells infected with HBV virus.

The HepG2 NTCP cell culture used in this experiment is capable of being able to support the full cycle of viral replication, being infected with the HBV virus.

The HBV preparation used in this experiment for infection of HepG2 / NTCP cells was obtained from supernatants of cell cultures of the AD38 line,

secreting HBV in the absence of tetracycline (Antimicrob. Agents Chemother. 1997 Aug; 41 (8): 1715-20).

HepG2 NTCP cells were plated in 96-well plates (3.0 x 10 cells per well) in Huh-7 medium (50 μl per well). For each test compound

6 solutions were prepared in Huh-7 medium in a concentration range of 0.2-60 μM. After 4 hours after cell sowing, the stock solution was removed by suction, after which 50 μl of a solution of the test compound, as well as 50 μl of HBV were added to each well. Thus, the final concentrations of the tested compounds were in the range of 0.1-30 μmol / L. After incubation for 24 hours, the viral medium was removed by aspiration, and test compounds at initial concentrations were added to the cultures to a final volume of 200 μl. Cells and test compounds are incubated for a further 7 days at 37 ° C in an air atmosphere containing 5% carbon dioxide (C0 _2). Cultural supernatants were tested for HBeAg content using the commercial HBeAg Elisa 4.0 diagnostic kit (MP Biomedicals, Singapore) in accordance with the manufacturer's recommendations. Briefly, 10 to 50 μl of the supernatants were added to 96-well plates with adsorbed anti-HBeAg antibodies, a second anti-HBeAg antibody labeled with horseradish peroxidase was added and incubated for 1 hour at room temperature. Wash each well three times with PBS, then treat with 100 μl / moon of OPD solution (prepared by dissolving 1 tablet of OPD in 12 ml of citrate-phosphate buffer with 5 μl of 30% H ₂ 0 ₂ ) for 30 minutes in the dark at room temperature. The reaction was stopped by adding 100 μl of 2N H ₂ S0 ₄ to each well, after which absorbance was measured at a wavelength of 490 nm. The value of EC ₅ about, which is the concentration of the test compound at which the virus replication is halved, was determined for each test compound using the Xlfit program.

The results indicate a high antiviral activity of inhibitors of General formula 1 against HBV. So, for example, in table 1

EC ₅ 0 values are presented for some representatives of inhibitors of general formula 1 with respect to HBV.

Table 1. Antiviral activity of inhibitors of general formula 1, against hepatitis B viruses (HBV) in human hepatoma cell culture (HepG2 / NTCP) infected with HBV in vitro.

Inhibitor

ECse, μΜ

JVa CAS JVa 1.3 CAS 9 1519-99-6 0.48

1.5 CAS 896681-30-40 0.17

1.7 CAS 951541-95-0 0.43

1.12 CAS 866846-93-7 0.67

1.13 CAS 896681-15-5 0.08

Example 2. Determination of the cytotoxicity of inhibitors of the General formula 1.

To determine the cytotoxicity of substances, HepG2 / NTCP cells were seeded in a black microplate with a transparent bottom (96 cells, 3.0 x 10 ³ cells per well). Three independent repeats were used for each inhibitor. Test inhibitors were added after 18 hours, after which the cells were incubated with substances for 7 days.

Wash twice each well with PBS phosphate-buffered saline (0.2 ml / moon) and then lyse the cells by adding cell buffer (50 μl / moon) (all of these reagents are included in the ATPLite kit, Perkin-Elmer, USA). The microplate was incubated for 5 minutes on a rotating platform at 600 rpm, after which 50 μl of substrate solution (part of the ATPLite kit) was added to each well. Incubated for another 5 minutes on a rotating platform at 600 rpm, kept for 10 minutes in the dark and then measured the luminescence on a TopCount NXT instrument (Packard, Perkin Elmer).

The CC50 value, which is the concentration of the test compound at which the cell viability is reduced by half, was determined for each

a test inhibitor of general formula 1 and general formula 2 using the Xlfit program. The results indicate a low cytotoxicity of inhibitors (CC50> 30 μΜ) of the general formula 1.

Example 3. Obtaining a drug in the form of tablets. 1600 mg of starch, 1600 mg of crushed lactose, 400 mg of talc and 1000 mg of inhibitor 1.13 are mixed and pressed into a block. The resulting bar is crushed into granules and sieved through sieves, collecting granules with a size of 14-16 mesh. The granules obtained are tabletted into a suitable tablet form weighing 560 mg each.

Example 4. Obtaining a drug in the form of capsules. Thoroughly mix the 1.13 inhibitor with lactose powder in a ratio of 2: 1. The resulting the powder mixture is packaged in 600 mg in a suitable size gelatin capsule.

Example 5. Obtaining a medicinal product in the form of injection compositions for intramuscular, intraperitoneal or subcutaneous injection. 500 mg of inhibitor 1.13 are mixed with 300 mg of chlorobutanol, 2 ml of propylene glycol and 100 ml of injection water. The resulting solution is filtered and placed in 1 ml ampoules, which are sealed.

Industrial applicability

The invention can be used in medicine and veterinary medicine.

Claims

Claim

1. The use of compounds representing 1, 1-dioxo-1, 4-dihydro-2H-benzo [1, 2,4] thiadiazin-3-ones of general formula 1, their pharmaceutically acceptable salts and / or hydrates as replication inhibitors hepatitis B virus

where Ar ¹ and Ag ² are phenyl optionally substituted with one, two or three identical or different substituents selected from C ^ C ^ alkyl, methoxy, halogen, carboxyl and carbonitrile.

2. The use of compounds according to claim 1, which are 1, 1-dioxo-1, 4-dihydro-2H-benzo [1, 2,4] thiadiazin-3-ones selected from a number of compounds of the formula 1.1-1.15

1.11 1.12 1.13 1.14 1.15

3. A pharmaceutical composition in the form of tablets, capsules or injections in a pharmaceutically acceptable package for the treatment of hepatitis B, comprising a hepatitis B virus replication inhibitor of general formula 1 according to claims 1, a pharmaceutically acceptable salt and / or hydrate thereof in a therapeutically effective amount.

4. A method of obtaining a pharmaceutical composition by mixing with an inert excipient and / or solvent of at least one inhibitor of general formula 1 according to claims. 1 or a pharmaceutically acceptable salt and / or hydrate thereof in a therapeutically effective amount.

5. A method for the prevention and treatment of hepatitis B virus is to introduce to the patient a hepatitis B virus replication inhibitor of general formula 1 according to claims. 1, its pharmaceutically acceptable salt and / or hydrate or pharmaceutical composition according to ππ. 3, containing as an active component at least one hepatitis B virus replication inhibitor of general formula 1 according to claim 1, a pharmaceutically acceptable salt and / or hydrate thereof.

6. A method of inhibiting the activity of hepatitis B virus in vivo, comprising the step of contacting an inhibitor of general formula 1 according to claims. 1, a pharmaceutically acceptable salt and / or hydrate and hepatitis B virus thereof.