EA044023B1 - SUBSTITUTED 3,4,12,12A-TETRAHYDRO-1H-[1,4]OXAZINO[3,4-C]PYRIDO[2,1-F][1,2,4]TRIAZINE-6,8-DIONE, PHARMACEUTICAL COMPOSITION, METHODS OF THEIR OBTAINING AND APPLICATION - Google Patents

Description

Field of technology

This invention relates to a previously unknown compound - substituted 3,4,12,12atetrahydro-1H-[1,4]oxazino[3,4-c]pyrido[2,1-4|[1,2,4]triazine-6 ,8-dione, which is an effective remedy for the prevention and treatment of viral diseases, including influenza.

Prior Art

Influenza is an acute infectious disease of the respiratory tract caused by the influenza virus. Included in the group of acute respiratory viral infections (ARVI). Periodically spreads in the form of epidemics and pandemics. Currently, more than 2000 variants of the influenza virus have been identified, differing in their antigenic spectrum. According to WHO estimates, from all variants of the virus during seasonal epidemics in the world, from 250 to 500 thousand people die annually (most of them are over 65 years old), in some years the number of deaths can reach a million.

The virus was first isolated in the 1930s. Influenza viruses belong to the orthomyxoviridae family - a family of RNA viruses. This family includes seven types of viruses, including influenza A virus, influenza B virus, influenza C virus, influenza D virus. The three types of influenza virus, which are identified by antigenic differences in their nucleoprotein and matrix protein, infect vertebrates as follows. Influenza A virus infects humans, other mammals, and birds and causes all influenza pandemics. Influenza B virus infects humans and seals. Influenza C virus infects humans, pigs and dogs. Influenza D virus infects (isolated in 2012) farm animals. As far as is known, influenza D does not infect human cells.

Viruses containing three subtypes of surface proteins hemagglutinin (HA) (H1, H2, H3) and two subtypes of neuraminidase (NA) (N1, N2) are of epidemic importance for humans. Influenza A and B viruses contain NA and HA as the main structural and antigenic components of the viral particle, which have hemagglutinating and neuraminidase activities. Influenza C virus does not have neuraminidase, but instead possesses a hemagglutinin esterase (entry) protein (HEF).

A significant achievement of modern biology and medicine is the development and implementation of methods for the treatment of viral infections, treatment aimed at eliminating the cause of the disease, based on the use of specific inhibitors that block the functional activity of the virus at different stages of its replication cycle.

Most anti-influenza drugs currently on the market are inhibitors of neuraminidase (zanamivir, oseltamivir, perimivir) or M2 proton ion channel protein (amantadine, rimantadine) [Hayden, F. G., Antivirals for influenza: historical perspectives and lessons learned, Antiviral. Res., 71, 372-8 (2006)]. However, these targets, especially the latter, are susceptible to rapid mutations that can confer antiviral resistance due to the inability of the viral RNA-dependent RNA polymerase (RdRp) to proofread RNA during replication. In fact, the World Health Organization's World Influenza Program has reported that >99% of seasonal influenza A strains are now resistant to amantadine and rimantadine [Barr, I.G. et al., WHO recommendations for the viruses used in the 2013-2014 Northern Hemisphere influenza vaccine: Epidemiology, antigenic and genetic characteristics of influenza A(H1N1) pdm09, A(H3N2) and B influenza viruses collected from October 2012 to January 2013, vaccine 32, 4713-25 (2014)]. This has led to the search for new antiviral compounds that target other important viral processes [Yen, H.L., Current and novel antiviral strategies for influenza infection, Curr. Opin. Virol., 18, 126-134 (2016)]. In this regard, the RNA-dependent RNA polymerase (RdRp) or RNA replicase of the influenza virus itself is an attractive drug target because it develops drug resistance relatively slowly, is conserved across genotypes, and is essential for viral replication.

In the last decade, understanding of the influenza virus RdRp has expanded dramatically due to the high-resolution elucidation of the architecture of the influenza virus endonuclease [Dias, A. et al., The cap-snatching endonuclease of influenza virus polymerase resides in the PA subunit, Nature, 2009, 458, 914 -918; Pflug, A. et al., Structure of influenza A polymerase bound to the viral RNA promoter, Nature, 2014, 516, 355-360.] and the discovery of the complete heterotrimeric structure of RdRp [Hengrung, N. et al., Crystal structure of the RNA-dependent RNA polymerase from influenza C virus, Nature, 2015, 527, 114-117].

Located in the nuclei of infected cells, the influenza virus RdRp is represented by a heterotrimer consisting of three subunits: PA, PB1 and PB2. RdRp is responsible for the replication and transcription of eight different RNA segments of the viral genome. This viral polymerase synthesizes viral mRNAs using short Cap primers from the host cell. These Cap primers are derived from cellular transcripts (RNA) using a unique cap-dependent mechanism. In an infected cell, the PB2 viral subunit binds the Cap pre-mRNAs at their 5' ends. After this, these CAP-RNAs associated with the PB2 subunit are cleaved by the viral endonuclease after 10-13 nucleotides. Biochemical and structural studies have shown that the endonuclease active center is located at the amino-terminal region of 209 amino acid residues of the PA subunit. This domain has endogenous RNA and DNA endonuclease activity, which is strongly activated by manganese ions. This is consistent with observations that reported a manganese-dependent endonuclease activity of an intact three-subunit polymerase [Dias, A. et al., 2009]. The possibility of inhibiting this endonuclease activity was first established almost a quarter of a century ago using the example of 4-substituted 2,4-dioxobutanoic acids (for example, compounds A1 A2) [J. Tomassini et al., Inhibition of cap (m7GpppXm)-dependent endonuclease of influenza vims by 4-substituted 2,4-dioxobutanoic acid compounds, Antimicrob. Agents Chemoter., 1994, 2827-2837].

Al A2

Somewhat later, natural inhibitors of Cap-dependent transcriptase of influenza A and B viruses were isolated from the culture of Delitschia confertaspora, representing substituted 2,6-diketopiperazines, including Flutimide (A3) and its analogues [J. Tomassini et al., A Novel Antiviral Agent Which Inhibits the Endonuclease of Influenza Viruses, Antimicrob. Agents Chemoter., 1996, 1189-1193], as well as synthetic drugs Favipiravir (A4) [Furuta Y. et al., T-705 (favipiravir) and related compounds: Novel broad-spectrum inhibitors of RNA viral infections. Antiviral Research. 2009, 82 (3), 95-102] and Pimodier (A5) [Clark, M.P. et al., Discovery of a novel, first-in-class, orally bioavailable azaindole inhibitor (VX-787) of influenza PB2, J. Med. Chem., 2014, 57, 6668-6678; Byrn, R.A. et al., Preclinical activity of VX-787, a first-in-class, orally bioavailable inhibitor of the influenza virus polymerase PB2 subunit, Antimicrob. Agents Chemother., 2015, 59, 1569-1582, https://ichgcp. net/clinical-trials-registry/NCT02342249].

Pimodivir, JNJ-3872, JNJ-872,

Flutimide Favipiiavh; T-705, Avigan JNJ-63623872, VX 78

A3 A4 A5

Flutimide Favipiravir Pimodivir

Favipiravir (A4), a broad-spectrum drug that targets multiple viral RdRps, including influenza virus, was approved in Japan in 2014 and the United States in 2015 for emergency use in the event of an influenza pandemic, despite some significant toxicity concerns this drug [Nagata, T. et al., Favipiravir: a new medication for the Ebola virus disease pandemic, Disaster. Med. Public Health Prep., 9, 79-81 (2015)].

Pimodivir (A5) is a first-in-class influenza virus replication inhibitor that blocks the PB2 activity of the influenza virus viral polymerase complex [Clark, M.P. et al., Discovery of a novel, first-in-class, orally bioavailable azaindole inhibitor (VX-787) of influenza PB2, J. Med. Chem., 2014, 57, 6668-6678; Byrn, R.A. et al., Preclinical activity of VX-787, a first-in-class, orally bioavailable inhibitor of the influenza virus polymerase PB2 subunit, Antimicrob. Agents Chemother., 2015, 59, 1569-1582, https://ichgcp.net/clinical-trials-registry/NCT02342249]. The drug is in a phase 3 clinical trial evaluating the efficacy and safety of pimodivir in combination with standard of care in adolescent, adult, and elderly patients with influenza A infection [https://clinicaltrials.gov/ct2/show/NCT033 76321].

Relatively recently, polyheterocyclic inhibitors of Cap-dependent endonuclease were patented, including a fragment of substituted 2,4-dioxobutanoic acid, including inhibitors A6, A7 [EP 2620436B1 (priority 2010), https://patents.google.com/patent/EP2620436B1] , A8, A9 [US 9827244B2 (priority 2016)] and A10, A11 [WO 2016175224A1; WO 2018030463 (priority 2016)].

- 2 044023

In this series of inhibitors, apparently the most effective is Baloxavir A10 (Baloxavir, Baloxavir acid, BCA, (12aR)-7-hydroxy-12-[(11S)-5,11-dihydro[1]benzothiepino[3, 4-b]pyridin-5-yl]3,4,12,12a-tetrahydro-1H-[1,4]oxazino[3,4-c]pyrido[2,1-1][1,2,4] triazine-6,8-dione), the prodrug of which Baloxavir marboxil A11 (Baloxavir marboxil, BXM, S-033188) - trade name Xofluza® - was approved in 2018 for the treatment of influenza A and B infections in Japan and the USA.

Xofluza is the first oral antiviral drug with a new mechanism of action for the treatment of influenza in almost 20 years [S. Omoto at al., Characterization of influenza virus variants induced by treatment with the endonuclease inhibitor baloxavir marboxil, Scientific Reports, 2018, 8, article number: 9633].

Given that influenza poses a serious threat to public health (globally, annual epidemics result in 3-5 million cases of severe illness, millions of hospitalizations and up to 650,000 deaths worldwide), the search for new anti-influenza drugs with improved characteristics seems appropriate.

The authors unexpectedly discovered that the previously unknown substituted 3,4,12,12a-tetrahydro-1H[1,4]oxazino[3,4-c]pyrido[2,1-f][1,2,4]triazine-6 ,8-dione of general formula 1

where R ¹ is 6,7-difluoro-5,10-dihydrothieno[3,2-c][2]benzothipin-10-yl, 7,8-difluoro-4,9-dihydrothieno[2,23c][2] benzothiepin-4-yl, (3,4-difluorophenyl)(phenyl)methyl, (3,4-difluorophenyl)(2-methylsulfanylphenyl)methyl, diphenylmethyl, (3,4-difluorophenyl)(2-methylsulfanylphenyl)methyl, diphenylmethyl, bis(4-fluorophenyl)methyl;

R ² - hydrogen or protective group,

- 3 044023 as well as its stereoisomer, prodrug thereof, pharmaceutically acceptable salt, solvate hydrate and crystalline or polycrystalline form thereof, including this new compound, pharmaceutical composition, are effective anti-influenza agents.

Below are definitions of various terms used to describe this invention. These definitions apply to the terms as used in this specification and claims unless otherwise limited in specific cases, either individually or as part of a larger group.

The term aryl as used herein, unless otherwise specified, refers to substituted or unsubstituted phenyl (Ph), biphenyl or naphthyl; preferably the term aryl refers to substituted or unsubstituted phenyl. The aryl group may be substituted by one or more moieties selected from hydroxyl, F, Cl, Br, I, amino, alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate, phosphonic acid, phosphate and phosphonate, optionally protected or not, known to specialists in the field, for example, such as those described in the book by Green T.V. and Vatsa P.G.M. Protective Groups in Organic Synthesis (T/W. Greene & P.G.M. Wuts, Protective Groups in Organic Synthesis, 3rd ed., John Wiley & Sons, 1999).

The term heteroaryl as used herein refers to a mono- or polycyclic aromatic radical in which one or more ring atoms are selected from S, O and N and the remaining ring atoms are carbon. Heteroaryl includes, but is not limited to, pyridinyl, pyrazinyl, pyrimidinyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzoxazolyl, or quinoxalinyl.

The term alkyl as used herein refers to saturated, straight or branched hydrocarbon radicals containing from one to six carbon atoms. Examples of C1- _C6 alkyl radicals include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl and tert-butyl.

The term protecting group as used herein refers to substituents attached to the oxygen of the alcohol group, typically used to block or protect the functionality of the alcohol, or other hydroxy compound, without interfering with the reaction with the compound of other functional groups [in Greene's Protective Groups in Organic Synthesis, editor Peter GM, chapter 2, Protection for the Hydroxyl Group, Including 1,2- and 1,3-Diols, p. 17-471, 2014, https://onlinelibrary.wiley.com/doi/book/10.1002/9781118905074]. Preferably selected from the range consisting of (C1-C ₃ -alkyl)oxycarbonyloxy, {[(C1-C ₃ -alkyl)oxycarbonyl]oxy}methoxy, {[2-(C1-C ₃ alkyl)oxyethoxy]carbonyl}oxy, ( {[(1R)-2-[(C ₁ -C ₃ -alkyl)oxy]-1-methylethoxy]}carbonyl)oxy, {[(3S)-tetrahydrofuran-3-yloxy]carbonyl}oxy, [(tetrahydro- 2H-pyran-4-yloxy)carbonyl]oxy, {[(1-acetylazetidin)-3-yloxy]carbonyl}oxy, {[(C1-C ₃ -alkyl)oxycarbonyl]oxy}methoxy, ({[2-( C1-C ₃ alkyl)oxyethoxy]carbonyl}oxy)methoxy.

The term prodrug refers to compounds of the invention that are cleaved chemically or metabolically to become, by solvolysis or under physiological conditions, a compound of the invention that is pharmaceutically active in vivo. Prodrugs often have higher solubility, histocompatibility, delivery or sustained release in mammals (Bungard, H., Design of products, pp. 7-9, 21-24, Elsevier, Amsterdam, 1985). Prodrugs include acid derivatives well known to those skilled in the art, such as, for example, esters prepared by reacting an acid parent compound with a suitable alcohol, or amides prepared by reacting an acid parent compound with a suitable amine. Examples of prodrugs include, but are not limited to, acetate, formate, benzoate or other acylated derivatives of alcohol or amine functional groups in the compounds of the present invention.

The term active ingredient (drug substance) refers to a physiologically active substance of synthetic or other (biotechnological, plant, animal, bactericidal, etc.) origin, possessing pharmacological activity, which is the active ingredient of a pharmaceutical composition.

The term medicinal product means a substance (or a mixture of substances in the form of a pharmaceutical composition) in the form of tablets, capsules, injections, ointments and other finished forms, intended to restore, correct or change physiological functions in humans and animals, as well as for the treatment and prevention of diseases , diagnostics, anesthesia, contraception, cosmetology and other things.

The term therapeutic cocktail represents a simultaneously administered combination of two or more drugs that have different mechanisms of pharmacological action and are aimed at different biotargets involved in the pathogenesis of the disease.

The term 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, auxiliary, dispensing and receiving agents, delivery vehicles, such as preservatives, stabilizers, fillers, disintegrants, humectants, emulsifiers, suspending agents, thickeners, sweeteners, flavoring agents, flavoring agents, antibacterial agents, fungicides, lubricants, sustained delivery regulators, the choice and ratio of which depends on the nature

- 4 044023 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, and mixtures of these substances. Protection against the action of microorganisms can be provided by a variety of antibacterial and antifungal agents, such as parabens, chlorobutanol, sorbic acid and the like. The composition may also include isotonic agents, for example, sugars, sodium chloride and the like. Prolonged action of the composition can be achieved using agents that slow down the absorption of the active component, for example, aluminum monostearate and gelatin. Examples of suitable carriers, solvents, diluents and delivery vehicles include water, ethanol, polyalcohols, as well as mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters (such as ethyl oleate). Examples of fillers are lactose, milk sugar, sodium citrate, calcium carbonate, calcium phosphate and the like. Examples of grinders and distributing agents are starch, alginic acid and its salts, and silicates. Examples of lubricants are magnesium stearate, sodium lauryl sulfate, talc, and high molecular weight polyethylene glycol. A pharmaceutical composition for oral, sublingual, transdermal, intramuscular, intravenous, subcutaneous, topical or rectal administration of an active ingredient, alone or in combination with another active ingredient, can be administered to animals and humans in a standard dosage form, mixed with conventional pharmaceutical carriers. Suitable unit dosage forms include oral forms such as tablets, gelatin capsules, pills, powders, granules, chewing gums and oral solutions or suspensions, sublingual and buccal administration forms, aerosols, implants, topical, transdermal, subcutaneous, intramuscular, intravenous, intranasal or intraocular administration forms and rectal administration forms.

The term excipient as used herein refers to a compound that is used to prepare a pharmaceutical composition and is generally safe, non-toxic and neither biologically nor otherwise undesirable, and includes excipients that are acceptable for use in veterinary medicine, as well as pharmacologically acceptable for human use. The compounds of this invention may be administered alone, but will typically be administered in mixtures with one or more pharmaceutically acceptable excipients, diluents or carriers selected based on the intended route of administration and standard pharmaceutical practice.

The term pharmaceutically acceptable salt means the relatively non-toxic organic and inorganic salts of the acids and bases claimed in the present invention. These salts can be obtained in situ during the synthesis, isolation or purification of compounds, or specially prepared. In particular, base salts can be prepared specifically starting from 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, S.M. 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, whereby metal and amine salts can be synthesized. Metallic 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 prepared include sodium hydroxide, carbonate, bicarbonate and hydride, potassium hydroxide and bicarbonate, potash, lithium hydroxide, calcium hydroxide, magnesium hydroxide, zinc hydroxide. The organic bases from which salts of the claimed acids can be obtained are amines and amino acids that are sufficiently basic to form a stable salt and suitable for use for medical purposes (in particular, they must 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, such as choline, tetramethylammonium, tetraethylammonium and the like, can be used for salt formation. The basic amino acids that can be used as amino acids are lysine, ornithine and arginine.

The term crystalline form means the structure of a substance characterized by the packing of its constituent molecules into one of the types of crystal lattice.

The term polycrystalline form means the structure of a substance having a polycrystalline structure, i.e. consisting of many small single crystals, i.e. crystallites of a certain crystalline form.

The term therapeutically effective amount as used herein means the amount of sub

- 5 044023 station, prodrug or drug necessary to reduce the symptoms of a disease in a subject. The dosage of a substance, prodrug or drug will be tailored to the individual requirements in each case. This dose may vary widely depending on numerous factors, such as the severity of the disease being treated, the age and general health of the patient, other drugs with which the patient is being treated, the route and form of administration and the experience of the attending physician. For oral administration, the daily dose is approximately 0.01 to 10 g, including all values in between, per day in monotherapy and/or combination therapy. The preferred daily dose is from about 0.1 to 7 g per day. Typically, treatment is started with a large initial loading dose to quickly reduce or eliminate the virus, followed by a tapering dose to a level sufficient to prevent a resurgence of infection.

The term solvate means a complex or aggregate formed by one or more molecules of a solute, i.e. a compound of the invention or a pharmaceutically acceptable salt thereof and one or more solvent molecules. Such solvates are typical solid crystals having an essentially fixed molar ratio of solute to solvent. Representative solvents include, but are not limited to, water, ethanol, isopropanol, acetic acid, etc. When the solvent is water, the solvate formed is a hydrate.

The term subject means a mammal, which includes, but is not limited to, cattle, pigs, sheep, chickens, turkeys, buffalo, llamas, ostriches, dogs, cats and humans, preferably the subject is a human. It is contemplated that the method of treating a subject may include any of the prodrugs of general formula 1, a stereoisomer thereof, an isotopically enriched analogue thereof, a pharmaceutically acceptable salt, hydrate, solvate, crystalline and polymorphic form thereof, or in combination thereof with another compound, including HCV NS5A inhibitor.

Disclosure of the Invention

The authors unexpectedly discovered that the previously unknown substituted 3,4,12,12a-tetrahydro-1H[1,4]oxazino[3,4-c]pyrido[2,1-f][1,2,4]triazine-6 ,8-dione of general formula 1

R ¹ I

⁰ O. 2 R where R ¹ is 6,7-difluoro-5,10-dihydrothieno[3,2-c][2]benzothipin-10-yl, 7,8-difluoro-4,9-dihydrothieno[2, 23c] [2]benzothiepin-4-yl, (3,4-difluorophenyl)(phenyl)methyl, (3,4-difluorophenyl)(2-methylsulfanylphenyl)methyl, diphenylmethyl, bis(4-fluorophenyl)methyl;

R ² - hydrogen or a protecting group selected from the series including benzyl, (C1-C ₃ -alkyl)oxycarbonyloxy, {[(C1-C ₃ -alkyl)oxycarbonyl]oxy} methoxy, {[2-(C1-C ₃ alkyl)oxyethoxy]carbonyl} oxy, ({(1 R)-2-[(C ₁ -C ₃ -αalkyl)oxy]-1 -methylethoxy]} carbonyl)oxy, {[(3 S)tetrahydrofuran-3 -yloxy ] carbonyl} oxy, [(tetrahydro-2H-pyran-4-yloxy)carbonyl]oxy, {[(1acetylazetidin)-3-yloxy]carbonyl}oxy, {[(C _g C ₃ -alkyl)oxycarbonyl]oxy}methoxy , ({[2-(C _g C ₃ alkyl)oxyethoxy]carbonyl}oxy)methoxy, as well as its stereoisomer, prodrug thereof, pharmaceutically acceptable salt, solvate hydrate and crystalline or polycrystalline form thereof, including this new compound, pharmaceutical composition, are effective anti-influenza drugs.

Preferred are (12aR)-12-(6,7-difluoro-5,10-dihydrothieno[3,2-c][2]benzothiepin-10-yl)-3,4,12,12atetrahydro-1R-[1 ,4]oxazino[3,4-c]pyrido[2,1-/|[1,2,4]triazine-6,8-dione of general formula 1.1, (12ai')-12-[(100)-6 ,7-difluoro-5,10-dihydrothieno[3,2-c][2]benzothipin-10-yl]-3,4,12,12atetrahydro-1R-[1,4]oxazino[3,4-c] pyrido[2,1-/|[1,2,4]triazine-6,8-dione of general formula 1.2, (12aA)-12-[(10A)-6,7-difluoro-5,10-dihydrothieno[ 3,2-c][2]benzothiepin-10-yl]-3,4,12,12atetrahydro-1R-[1,4]oxazino[3,4-c]pyrido[2,1-/|[1, 2,4]triazine-6,8-dione of general formula 1.3, stereoisomer thereof, prodrug thereof, pharmaceutically acceptable salt, solvate, hydrate and crystalline or polycrystalline form thereof

- 6 044023

where R ² has the above meaning.

More preferred are (12a/?)-7-benzyloxy-12-(6,7-difluoro-5,10-dihydrothieno[3,2-c][2]benzothipin-10-yl)3,4,12, 12a-tetrahydro-177-[1,4]oxazino[3,4-c]pyrido[2,1-/|[1,2,4]triazine-6,8-dione (1.1.1), (12a5) -7-benzyloxy-12-[(105)-6,7-difluoro-5,10-dihydrothieno[3,2-c][2]benzothipin-10yl]-3,4,12,12a-tetrahydro-1R -[1,4]oxazino[3,4-c]pyrido[2.1 _t /][1,2,4]triazine-6,8-dione (1.2.1), (12a5)-7-benzyloxy- 12-[(105)-6,7-difluoro-5,10-dihydrothieno[3,2-c][2]benzothipin-10yl]-3,4,12,12a-tetrahydro-III-[1,4] Oxazino[3,4-c]pyrido[2.1 _t /][1,2,4]triazine-6,8-dione (1.3.1), (12a5)-7-hydroxy-12-(6,7 -difluoro-5,10-dihydrothieno[3,2-c][2]benzothiepin-10-yl)3,4,12,12a-tetrahydro-1R-[1,4]oxazino[3,4-c]pyrido [2,1 _: /][1,2,4]triazine-6,8-dione (1.1.2), (12a5)-7-hydroxy-12-[(105)-6,7-difluoro-5, 10-dihydrothieno[3,2-c][2]benzothiepin-10yl]-3,4,12,12a-tetrahydro-1/7-[1,4]oxazino[3,4-c]pyrido[2,1 _: /][1,2,4]triazine-6,8-dione (1.2.2), (12а7?)-7-hydroxy-12-[(10А)-6,7-difluoro-5,10-dihydrothieno [3,2-c][2]benzothiepin-10yl]-3,4,12,12a-tetrahydro-177-[1,4]oxazino[3,4-c]pyrido[2,1 _: /][1 ,2,4]triazine-6,8-dione (1.3.2), (12a5)-12-(6,7-difluoro-5,10-dihydrothieno[3,2-c] [2]benzothiepine-10- yl)-6,8-dioxo3,4,12,12a-tetrahydro-1R-[1,4]oxazino[3,4-c]pyrido[2,1 _: /][1,2,4] ^t Riazin- 7-yl methyl carbonate (1.1.3), (12а7?)-12-[(105)-6,7-difluoro-5,10-dihydrothieno[3,2-с][2]benzothipin-10-yl] -6,8-dioxo3,4,12,12a-tetrahydro-1R-[1,4]oxazino[3,4-c]pyrido[2,17/][1,2,4]triazin-7-yl methyl carbonate (1.2.3), (12a5)-12-[(10^)-6,7-difluoro-5,10-dihydrothieno[3,2-c][2]benzothipin-10-yl]-6,8dioxo -3,4,12,12a-tetrahydro-1R-[1,4]oxazino[3,4-c]pyrido[2,1-[1,2,4]triazin-7-yl methyl carbonate (1.3 .3), {[(12a5)-12-(6,7-difluoro-5,10-dihydrothieno[3,2-c][2]benzothipin-10-yl)-6,8-dioxo3,4,12 ,12a-tetrahydro-1/7-[1,4]oxazino[3,4-c]pyrido[2,1^1(1,2,4]triazin-7-yl]oxy} methyl methyl carbonate (1.1. 4), ({(12aA)-12-[(105)-6,7-difluoro-5,10-dihydrothieno[3,2-c][2]benzothipin-10-yl]-6,8dioxo-3, 4,12,12a-tetrahydro-1R-[1,4]oxazino[3,4-c]pyrido[2,1-[1,2,4]triazin-7yl}oxy)methyl methyl carbonate (1.2. 4), ({(12aA)-12-[(10L)-6,7-difluoro-5,10-dihydrothieno[3,2-c][2]benzothipin-10-yl]-6,8dioxo-3 ,4,12,12a-tetrahydro-1R-[1,4]oxazino[3,4-c]pyrido[2,1-[1,2,4]triazin-7yl}oxy)methyl methyl carbonate (1.3 .4), their stereoisomer, their prodrug, pharmaceutically acceptable salt, solvate, hydrate and crystalline or polycrystalline form thereof

- 7 044023

Preferred are (12а7?)-12-(7,8-difluoro-4,9-dihydrothieno[2,3-с][2]benzothiepin-4-yl)-3,4,12,12atetrahydro-1//- [1,4]oxazino[3,4-c]pyrido[2,1/][1,2,4]triazine-6,8-dione of general formula 1.4, (12aA*)-12-[(105)- 7,8-difluoro-4,9-dihydrothieno[2,3-c|[2]benzothipin-4-yl]-3,4,12,12atetrahydro-1R-[1,4]oxazino[3,4-c ]pyrido[2,1/][1,2,4]triazine-6,8-dione of general formula 1.5, (12aR)-12-[(10L)-7,8-difluoro-4,9-dihydrothieno[ 2,3-c][2]benzothiepin-4-yl]-3,4,12,12atetrahydro-177-[1,4]oxazino[3,4-c]pyrido[2,1/][1,2 ,4] triazine-6,8-dione of general formula 1.6, stereoisomer thereof, prodrug thereof, pharmaceutically acceptable salt, solvate, hydrate and crystalline or polycrystalline form thereof

- 8 044023

OO., O CL, OO., ⁴ R Υ ² Υ

1.4 1.5 1.6 where R ² has the above meaning.

More preferred are (12a/?)-7-benzyloxy-12-(7,8-difluoro-4,9-dihydrothieno[2,3-c][2]benzothipin-4-yl)3,4,12, 12a-tetrahydro-\H-[1,4]oxazino[3,4-c]pyrido[2,1-f\[1,2,4]triazin-6,8-dione 1.4.1, (12aL )-7-benzyloxy-12-[(105)-7,8-difluoro-4,9-dihydrothieno[2,3-c][2]benzothipin-4yl]-3,4,12,12a-tetrahydro- 17/-[1,4]oxazino[3,4-c]pyrido[2,17][1,2,4]triazine-6,8-dione 1.5.1, (12a5)-7-benzyloxy-12- [(10L)-7,8-difluoro-4,9-dihydrothieno[2,3-c] [2]benzothipin-4yl]-3,4,12,12a-tetrahydro-1R-[1,4]oxazino [3,4-c]pyrido[2,1t][1,2,4]triazine-6,8-dione 1.6.1, (12a10-7-hydroxy-12-(7,8-difluoro-4, 9-dihydrothieno[2,3-c][2]benzothipin-4-yl)3,4,12,12a-tetrahydro-1R-[1,4]oxazino [3,4-c]pyri to[2,1 -f\[ 1,2,4]triazin-6,8-dione 1.4.2, (12а7?)-7-hydroxy-12-[(105)-7,8-difluoro-4,9-dihydrothieno[ 2,3-c][2]benzothiepin-4-yl]3,4,12,12a-tetrahydro-1/G-[1,4]oxazino[3,4-c]pyrido[2,17/][ 1,2,4]triazine-6,8-dione 1.5.2, (12a5)-7-hydroxy-12-[(10L)-7,8-difluoro-4,9-dihydrothieno[2,3-c] [2]benzothipin-4-yl]3,4,12,12a-tetrahydro-\H-[1,4]oxazino[3,4-c]pyrido[2,17][1,2,4]triazine -6,8-dione 1.6.2, (12a5)-12-(7,8-difluoro-4,9-dihydrothieno[2,3-c][2]benzothiepin-4-yl)-6,8- dioxo3,4,12,12a-tetrahydro-1/7-[1,4]oxazino[3,4-c]pyrido[2.1 _t /][1,2,4]triazin-7-yl methyl carbonate 1.4 .3, (12aL)-12-[(105)-7,8-difluoro-4,9-dihydrothieno[2,3-c][2]benzothipin-4-yl]-6,8-dioxo3,4, 12,12a-tetrahydro-177-[1,4]oxazino[3,4-c]pyrido[2,1 _: /][1,2,4]triazin-7-yl methyl carbonate 1.5.3, (12a5) -12-[( 10R)- 7,8-difluoro-4,9-dihydrothieno[2,3-c][2]benzothipin-4-yl]-6,8-dioxo3,4,12,12a-tetrahydro- 1R-[1,4]oxazino[3,4-c]pyrido[2,1-y][1,2,4]triazin-7-yl methyl carbonate 1.6.3, {[(12a7?)-12- (7,8-difluoro-4,9-dihydrothieno[2,3-c][2]benzothipin-4-yl)-6,8-dioxo3,4,12,12a-tetrahydro-1R-[1,4] Oxazino[3,4-c]pyrido[2,17/][1,2,4]triazin-7-yl]oxy}methyl methyl carbonate 1.4.4, {[(12a7?)-12-[(105) -7,8-difluoro-4,9-dihydrothieno[2,3-c][2]benzothipin-4-yl]-6,8-dioxo3,4,12,12a-tetrahydro-1/G-[1 ,4]oxazino[3,4-c]pyrido[2,1 _: /][1,2,4]triazin-7-yl]oxy}methyl methyl carbonate 1.5.4, {[(12aA)-12-[ (10L)- 7,8-difluoro-4,9-dihydrothieno[2,3-c][2]benzothipin-4-yl]-6,8-dioxo3,4,12,12a-tetrahydro-1/G- [1,4]oxazino[3,4-c]pyrido[2,1 _: /][1,2,4]triazin-7-yl]oxy}methyl methyl carbonate 1.6.4, its stereoisomer, their prodrug, pharmaceutical acceptable salt, solvate, hydrate and crystalline or polycrystalline form thereof

- 9 044023

Preferred are (12a/?)-12-[(3,4-difluorophenyl) (phenyl)methyl]-3,4,12,12a-tetrahydro-1R[1,4]oxazino[3,4-c]pyrido[ 2,1 _: /][1,2,4]triazine-6,8-dione of general formula 1.7, (12a/?)-12-[(/?)-(3,4-difluorophenyl)(phenyl)methyl] -3,4,12,12a-tetrahydro-1R[1,4]oxazino[3,4-c]pyrido[2,1 _: /][1,2,4]triazine-6,8-dione of general formula 1.8 , (12a/?)-12-[(5)-(3,4-difluorophenyl) (phenyl)methyl]-3,4,12,12a-tetrahydro-\H[1,4]oxazino[3, 4-c]pyrido[2,17/][1,2,4]triazine-6,8-dione of general formula 1.9, stereoisomer thereof, prodrug thereof, pharmaceutically acceptable salt, solvate, hydrate and crystalline or polycrystalline form thereof

- 10 044023

where R ² has the above meaning.

More preferred are (12a7?)-7-benzyloxy-12-[(3,4-difluorophenyl)(phenyl)methyl]-3,4,12,12a-tetrahydro-1R[1,4]oxazino[3,4- c]pyrido[2,1 -f\[ 1,2,4]triazine-6,8-dione (1.7.1), (12а7?)-7-benzyloxy-12-[(7?)-(3, 4-difluorophenyl)(phenyl)methyl]-3,4,12,12a-tetrahydro1R-[1,4]oxazino[3,4-c]pyrido[2,17/][1,2,4]triazine-6 ,8-dione (1.8.1), (12aA)-7-benzyloxy-12-[(5)-(3,4-difluorophenyl)(phenyl)methyl]-3,4,12,12a-tetrahydro1R-[1 ,4]oxazino[3,4-c]pyrido[2,1 _: /][1,2,4]triazine-6,8-dione (1.9.1), (12a7?)-7-hydroxy-12- [(3,4-difluorophenyl)(phenyl)methyl]-3,4,12,12a-tetrahydro-1R[1,4]oxazino[3,4-c]pyrido[2,1-f\[1,2 ,4]triazine-6,8-dione (1.7.2), (12а/?)-7-hydroxy-12-[(/?)-(3,4-difluorophenyl)(phenyl)methyl]-3,4 ,12,12a-tetrahydro-III[1,4]oxazino[3,4-c]pyrido[2,1 /][1,2,4]triazine-6,8-dione (1.8.2), (12aA )-7-hydroxy-12-[(5)-(3,4-difluorophenyl)(phenyl)methyl]-3,4,12,12a-tetrahydro-1R[1,4]oxazino[3,4-c] pyrido[2,1 y][1,2,4]triazine-6,8-dione (1.9.2), (12а/?)-12-[(3,4-difluorophenyl)(phenyl)methyl] -6 ,8-dioxo-3,4,12,12a-tetrahydro-1R[1,4]oxazino[3,4-c]pyrido[2,1 _: /][1,2,4]triazin-7-yl methyl carbonate (1.7.3), (12a7?)-12-[(/?)-(3,4-difluorophenyl)(phenyl)methyl]-6,8-dioxo-3,4,12,12a-tetrahydro-1R [1,4]oxazino[3,4-c]pyrido[2,1 _: /][1,2,4]triazin-7-yl methyl carbonate (1.8.3), (12a7?)-12-[( b)-(3,4-difluorophenyl)(phenyl)methyl]-3,4,12,12a-tetrahydro-1R[1,4]oxazino[3,4-c]pyrido[2,1/][1, 2,4]triazin-7-yl methyl carbonate (1.9.3), ({(12а/?)-12-[(3,4-difluorophenyl)(phenyl)methyl]-6,8-dioxo-3,4 ,12,12a-tetrahydro-1R[1,4]oxazino[3,4-c]pyrido[2,1-[1,2,4]triazin-7-yl}oxy)methyl methyl carbonate (1.7. 4), ({(12a/?)-12-[(A)-(3,4-difluorophenyl)(phenyl)methyl]-6,8-dioxo-3,4,12,12a-tetrahydro-1R[1 ,4]oxazino[3,4-c]pyrido[2,1 _: /][1,2,4]triazin-7-yl}oxy)methyl methyl carbonate (1.8.4), ({(12a7?)- 12-[(b)-(3,4-difluorophenyl) (phenyl)methyl]-3,4,12,12a-tetrahydro-1R

[l,4]oKca3HHo[3,4-c]nHpHflo[2,l/|[l,2,4]TpHa3HH-7-HH}oKCH)MeTHH methyl carbonate (1.9.4), its stereoisomer, their prodrug, pharmaceutically acceptable salt, solvate, hydrate and crystalline or polycrystalline form thereof

- 11 044023

Preferred are (12aL)-12-[(3,4-difluorophenyl)(2-methylsulfanylphenyl)methyl]-3,4,12,12a-tetrahydro1H-[1,4]oxazino[3,4-c' ]pyrido[2,1 _: /][1,2,4]triazine-6,8-dione of general formula 1.10, (12a7?)-12-[(A)-(3,4-difluorophenyl)(2-methylsulfanylphenyl )methyl]-3,4,12,12atetrahydro-1R-[1,4]oxazino[3,4-c]pyrido[2,1-[1,2,4]triazine-6,8-dione total formula 1.11, (12a7?)-12-[(5)-(3,4-difluorophenyl)(2-methylsulfanylphenyl)methyl]-3,4,12,12atetrahydro-1R-[1,4]oxazino[3,4 -c]pyrido[2,1-/|[1,2,4]triazine-6,8-dione of general formula 1.12, stereoisomer thereof, prodrug thereof, pharmaceutically acceptable salt, solvate, hydrate and crystalline or polycrystalline form thereof

- 12 044023

1.10 1.11 1.12 where R ² has the above meaning.

More preferred are (12aL)-7-benzyloxy-12-[(3,4-difluorophenyl)(2-methylsulfanylphenyl)methyl]3,4,12,12a-tetrahydro-1R-[1,4]oxazino[3,4 -c]pyrido[2,1 -f\[1,2,4]triazine-6,8-dione (1.10.1), (12aA)-7-benzyloxy-12-[(A)-(3,4 -difluorophenyl)(2-methylsulfanylphenyl)methyl]3,4,12,12a-tetrahydro-1//-[1,4]oxazino[3,4-c]pyrido[2,1 ₇ /][1,2, 4]triazine-6,8-dione (1.11.1), (12a7?)-7-benzyloxy-12-[(0)-(3,4-difluorophenyl)(2-methylsulfanylphenyl)methyl]3,4,12 ,12a-tetrahydro-1//-[1,4]oxazino[3,4-c]pyrido[2,1 ₇ /][1,2,4]triazine-6,8-dione (1.12.1), (12aL)-7-hydroxy-12-[(3,4-difluorophenyl)(2-methylsulfanylphenyl)methyl]-3,4,12,12atetrahydro-1R-[1,4]oxazino[3,4-c]pyrido [2,1-y][1,2,4]triazine-6,8-dione (1.10.2), (12aA)-7-hydroxy-12-[(A)-(3,4-difluorophenyl)( 2-methylsulfanylphenyl)methyl]3,4,12,12a-tetrahydro-1R-[1,4]oxazino[3,4-c]pyrido[2,1t][1,2,4]triazine-6,8 -dione (1.11.2), (12a7?)-7-hydroxy-12-[(5)-(3,4-difluorophenyl)(2-methylsulfanylphenyl)methyl]3,4,12,12a-tetrahydro-1R- [1,4]oxazino[3,4-c]pyrido[2,1-y][1,2,4]triazine-6,8-dione (1.12.2), (12aL)-12-[(3 ,4-difluorophenyl)(2-methylsulfanylphenyl)methyl]-6,8-dioxo-3,4,12,12atetrahydro-1R-[1,4]oxazino[3,4-c]pyrido[2,17/][ 1,2,4]triazin-7-yl methyl carbonate (1.10.3), (12a/?)-12-[(A)-(3,4-difluorophenyl)(2-methylsulfanylphenyl)methyl]-6,8 -dioxo3,4,12,12a-tetrahydro-1R-[1,4]oxazino[3,4-c]pyrido[2,1 _: /][1,2,4]triazin-7-yl methyl carbonate (1.11 .3), (12aL)-12-[(5)-(3,4-difluorophenyl)(2-methylsulfanylphenyl)methyl]-3,4,12,12atetrahydro-1R-[1,4]oxazino[3,4 -c]pyrido[2,1-y][1,2,4]triazin-7-yl methyl carbonate (1.12.3), ({(12aA)-12-[(3,4-difluorophenyl)(2- methylsulfanylphenyl)methyl]-6,8-dioxo-3,4,12,12atetrahydro-1R-[1,4]oxazino[3,4-c]pyrido[2,1-/|[1,2,4]triazine -7-yl}oxy)methyl methyl carbonate (1.10.4), ({(12a7?)-12-[(L)-(3,4-difluorophenyl)(2-methylsulfanylphenyl)methyl]-6,8-dioxo3 ,4,12,12a-tetrahydro-1/7-[1,4]oxazino[3,4-c]pyrido[2,1- _: /][1,2,4]triazin-7-yl}oxy) methyl methyl carbonate (1.11.4), ({(12aA)-12-[($)-(3,4-difluorophenyl)(2-methylsulfanylphenyl)methyl]-3,4,12,12atetrahydro-1R-[1, 4]oxazino[3,4-c]pyrido[2,1-/|[1,2,4]triazin-7-yl}oxy)methyl methyl carbonate (1.12.4), stereoisomer thereof, prodrug thereof, pharmaceutically acceptable salt, solvate, hydrate and their crystalline or polycrystalline form

- 13 044023

Preferred is (12aR)-12-diphenylmethyl-3,4,12,12a-tetrahydro-1H-[1,4]oxazino[3,4c]pyrido[2,1-1][1,2,4]triazine- 6,8-dione of general formula 1.13, stereoisomer thereof, prodrug thereof, pharmaceutically acceptable salt, solvate, hydrate and crystalline or polycrystalline form thereof

where R ² has the above meaning.

- 14 044023

More preferred are (12aL)-7-benzyloxy-12-diphenylmethyl-3,4,12,12a-tetrahydro-1R-[1,4]oxazino[3,4c]pyrido[2,1t][1,2, 4]triazine-6,8-dione (1.13.1), (12а7?)-7-hydroxy-12-diphenylmethyl-3,4,12,12а-tetrahydro-177-[1,4]oxazino[3,4с ]pyrido[2,17][1,2,4]triazine-6,8-dione (1.13.2), (12aL)-12-diphenylmethyl-6,8-dioxo-3,4,12,12a-tetrahydro -1/7-[1,4]oxazino[3,4с]pyrido[2,1t/][1,2,4]triazin-7-yl methyl carbonate (1.13.3), ({(12а/?) -12-diphenylmethyl-3,4,12,12a-tetrahydro-17/-[1,4]oxazino[3,4-c]pyrido[2,1/|[1,2,4]triazin-7-yl }oxy)methyl methyl carbonate (1.13.4), its stereoisomer, its prodrug, pharmaceutically acceptable salt, solvate, hydrate and crystalline or polycrystalline form thereof

Preferred are (12aR)-12-[bis(4-fluorophenyl)methyl]-3,4,12,12a-tetrahydro-1H[1,4]oxazino[3,4-c]pyrido[2,1-1] [1,2,4]triazine-6,8-dione of general formula 1.14, stereoisomer thereof, prodrug thereof, pharmaceutically acceptable salt, solvate, hydrate and crystalline or polycrystalline form thereof

where R ² has the above meaning.

More preferred are (12a7?)-7-benzyloxy-12-[bis(4-fluorophenyl)methyl]-3,4,12,12a-tetrahydro-177[1,4]oxazino[3,4-c]pyrido[ 2,1 -f\[ 1,2,4]triazine-6,8-dione (1.14.1), (12a7?)-7-hydroxy-12-[bis(4-fluorophenyl) methyl]-3,4 ,12,12a-tetrahydro-1R[1,4]oxazino[3,4-c]pyrido[2,1-U][1,2,4]triazine-6,8-dione (1.14.2), ( 12а7?)-12-[bis(4-fluorophenyl) methyl]-6,8-dioxo-3,4,12,12а-tetrahydro-177[1,4]oxazino[3,4-с]pyrido[2, 1;/][1,2,4]triazin-7-yl methyl carbonate (1.14.3), ({(12aL)-12-[bis(4-fluorophenyl)methyl]-3,4,12,12a- tetrahydro-1R-[1,4]oxazino[3,4c]pyrido[2,1 _: /][1,2,4]triazin-7-yl}oxy)methyl methyl carbonate (1.14.4), its stereoisomer, their prodrug, pharmaceutically acceptable salt, solvate, hydrate and crystalline or polycrystalline form thereof

- 15 044023

The subject of this invention is also a method for the preparation of compounds of general formula 1, which includes the preparation of 7-benzyloxy derivatives 1.1.1-1.14.1 by the reaction of (12aR)-7(benzyloxy)-3,4,12,12a-tetrahydro-1 H-[ 1 ,4]oxazino[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8-dione (2) respectively with 6,7-difluoro-5,10-dihydrothieno[ 3,2-c][2]benzothipin-10-ol (3), or 7,8-difluoro-7,8dihydrothieno[2,3-c][2]benzothipin-4-ol (4), or (3 ,4-difluorophenyl)(phenyl)methanol (5), (3,4difluorophenyl)(2-methylsulfanylphenyl)methanol (6), diphenylmethanol (7) or bis(4fluorophenyl)methanol (8)

The 7-benzyloxy derivatives 1.1.1, 1.4.1, 1.7.1 and 1.10.1 formed as a result of this reaction are separated into the corresponding diastereoisomers 1.2.1, 1.3.1, 1.5.1, 1.6.1, 1.8.1, 1.9.1 , 1.11.1 1.12.1 whose structure is confirmed by X-ray diffraction analysis (XRD) data. For example, the diastereomer (12aR)-7-(benzyloxy)-12-[(10S)-5,10-dihydrothieno[3,2-c][2]benzothipin-10-yl]-3,4,12, 12a-tetrahydro1H-[1,4]oxazino[3,4-c]nirido[2,1-f][1,2,4]triazine-6,8-dione (1.2.1) was recrystallized from ethanol and isopropanol , as a result of which orthorhombic crystals were obtained, which, according to X-ray diffraction data, are the corresponding solvates with ethanol 1.2.1-C ₂ H ₅ OH and isopropanol 1.2.1 h-C ₃ H ₇ OH in ratios 4:3 and 8:3 are presented in fig. 1 and 2.

Diastereomer (12aR)-7-benzyloxy-12-[(S)-(3,4-difluorophenyl)(phenyl)methyl]-3,4,12,12a-tetrahydro1H-[1,4]oxazino[3,4- c]pyrido[2,1-f][1,2,4]triazine-6,8-dione (1-9.1) was recrystallized from a mixture of dichloromethane-hexane, resulting in crystals obtained, which, according to X-ray diffraction analysis, were classified as to the triclinic crystal system (Fig. 3).

7-Benzyloxy derivatives 1.1.1-1.14.1 are debenzylated by heating in dimethyl sulfoxide in the presence of lithium chloride into the corresponding 7-hydroxy derivatives 1.1.2-1.14.2, and the latter are converted into prodrugs 1.1.3-1.14.3 and 1.1.4-1.14 .4. The starting material 6,7-difluoro-5,10-dihydrothieno[3,2c][2]benzothipin-10-ol (3) is a new compound, therefore this compound and the method for its preparation are also the subject of the present invention. According to the invention, the method for producing 6,7-difluoro-5,10-dihydrothieno[3,2-c][2]benzothipin-10-ol (3) involves the cyclization of 3,4-difluoro-2-[(3thienylthio)methyl]- benzoic acid (3.6) in the presence of PCl ₅ and reduction of the resulting 6,7-difluoro-5,10-dihydrothieno[3,2-c][2]benzothipin-10(5H)-one (3.7) with sodium borohydride (Scheme 1) .

- 16 044023

Scheme 1. Synthesis of 6,7-difluoro-5,10-dihydrothieno[3,2-c][2]benzothipin-10-ol (3).

The starting material 7,8-difluoro-7,8-dihydrothieno[2,3-c][2]benzothipin-10-ol (4) is a new compound, therefore this compound and the method for its preparation are also the subject of the present invention.

According to the invention, the method for producing 7,8-difluoro-7,8-dihydrothieno[2,3-c][2]benzothipin-10-ol (4) involves the cyclization of 3,4-difluoro-2-[(3-thienylthio)methyl ]-benzoic acid (4.3) in the presence of PCl ₅ and reduction of the resulting 6,7-difluoro-5,10-dihydrothieno[3,2-c][2]benzothiepin-10(5H)-one (4.4) with sodium borohydride (scheme 2).

Scheme 2. Synthesis of 7,8-difluoro-7,8-dihydrothieno[2,3-c][2]benzothipin-10-ol (4).

The original (3,4-difluorophenyl)(phenyl)methanol (5) is a new compound, therefore this compound and the method for its preparation are also the subject of the present invention.

According to the invention, the method for producing (3,4-difluorophenyl)(phenyl)methanol (5) involves the reduction of 3,4-difluorobenzophenone (5.1) with sodium borohydride (Scheme 3).

Scheme 3. Synthesis of (3,4-difluorophenyl)(phenyl)methanol (5).

The starting material (3,4-difluorophenyl)(2-methylsulfanylphenyl)methanol (6) is a new compound, therefore this compound and the method for its preparation are also the subject of the present invention. According to the invention, the method for producing (3,4-difluorophenyl)(2-methylsulfanylphenyl)methanol (6) consists of reducing (3,4-difluorophenyl)[2-methylsulfanyl)phenyl]methanol (6.6) with sodium borohydride (Scheme 4).

- 17 044023

Scheme 4. Synthesis of (3,4-difluorophenyl)(2-methylsulfanylphenyl)methanol (6).

NaBH ₄

MeOH rt, 30 min ^Br Mg

New 7-hydroxy-3,4,12,12a-tetrahydro-1H-[1,4]oxazino[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8diones , containing diarylmethyl substituents at position 12, unlike Baloxavir (BCA), are orally available influenza virus inhibitors with fairly high bioavailability. At the same time, Cmax and AUC of inhibitors are 1-2 orders of magnitude better than these VCA parameters.

Table 1

Pharmacokinetic parameters in plasma at RO mice of drugs at a dose of 15 mg/kg

A drug 1.8.2 1.9.2 1.13.2 1.14.4 VHA Control 1.8.2 1.9.2 1.13.2 1.14.4 VHA Kel, 1/h 0.25 0.27 0.24 0.27 0.19 T1/2, h 2.75 2.56 2.91 2.55 3.74 Tmax, h 1 1 0.5 2 4 Smax, ng/ml 2090 2375 684 308 11.9 AISlast, h ng/ml 9448 10958 2.657 1.822 80 AUCINF, h ng/ml 9469 10977 2,661 2,069 95 MITposl, h 3.48 3.34 2.64 3.23 4.79 RdiSposl, % nd* ND* 42 23 ND*

Kel - elimination constant.

T1/2 - half-life.

AUClast is the area under the concentration-time curve from the moment of drug administration to the determination of the last quantifiable concentration.

AUCINF is the area under the pharmacokinetic curve (concentration versus time) from 0 to infinity.

MRTafter - the average retention time in an unchanged form in the body from the moment of drug administration until the determination of the last quantifiable concentration.

Pharmacokinetic parameters, in particular, Cmax and AUClast in plasma upon RO administration of new proinhibitors to mice (Table 2), are 10-40 times higher than those of Baloxavir marboxil (BCM).

- 18 044023

table 2

Pharmacokinetic parameters in plasma during PO in mice of drugs at a dose of 15 mg/kg

A drug 1.3.4 1.8.4 1.9.4 1.13.4 1.14.4 5101 Control 1.3.2 1.8.2 1.9.2 1.13.2 1.14.4 5107 Kei, 1/h 0.25 0.25 0.26 0.30 0.74 0.17 T1/2, h 2.74 2.75 2.71 2.28 0.93 4.05 Tmax, h 2 1 1 2 4 4 Smack, ng/ml 48.9 2090 397 875 647 55.3 АiСsl, h ng/ml 224 9448 2078 3935 3186 392 AUCINF, h ng/ml 233 9469 2082 3938 3189 397 MKTposl, h 3.78 3.48 3.78 3.35 3.76 5.55 EdisPosl, % 23.9 62.2 39.6

7-Hydroxy derivatives 1.1.2-1.14.2 are nanomolar inhibitors of influenza virus isolates. For example, the activity of inhibitors 1.8.2, 1.9.2, 1.13.2 and 1.14.2 against influenza isolates

A/California/2009 (H1N1) in MDCK cell culture has an EC ₅₀ value of 2^4 nM, which practically coincides with this activity of Baloxavir (according to our data, EC ₅₀ = 2.4 nM). Inhibitor 1.3.2 against influenza isolates A/Califorma/2009 (H1N1), A/Aichi/2/69 (H3N2) and A/Perth/265/2009 (H1N1pdm09) (H275Y) in MDCK cell culture is even higher under identical conditions activity of Baloxavir (BCA) (Table 3).

Table 3

Antiviral activity of the new inhibitor 1.2.2 and BCA against influenza A virus isolates in MDCK cell culture

A drug A/Califorma/2009 (H1N1) A/Aichi/2/68 (H3N2) A/Perth/265/2009 (HlNlpdmO9) (H275Y) EC ₅ o, nM 1.3.2 <0.3 0.23 0.8 VHA 2.4 1.04 1.1

The subject of the present invention is the use of 7,8-difluoro-5,10-dihydrothieno[3,2c][2]benzothipin-10-ol (3), 7,8-difluoro-7,8-dihydrothieno[2,3-c ][2]benzothipin-10-ol (4), (3,4difluorophenyl)(phenyl)methanol (5), (3,4-difluorophenyl)(2-methylsulfanylphenyl)methanol (6), diphenylmethanol (7) and bis( 4-fluorophenyl)methanol (8) as an intermediate in the synthesis of compounds of general formula 1.

The subject of the present invention is the use of 7-benzyloxy derivatives 1.1.1-1.14.1 as an intermediate in the synthesis of the corresponding 7-hydroxy derivatives 1.1.2-1.14.2.

The subject of the present invention is the use of 7-hydroxy derivatives 1.1.2-1.14.2 as an intermediate in the synthesis of prodrugs 1.1.3-1.14.3 and 1.1.4-1.14.4.

The subject of the present invention is the use of 7-hydroxy derivatives 1.1.2-1.14.2, or stereoisomers thereof, pharmaceutically acceptable salt, solvate, hydrate and crystalline or polycrystalline form thereof, as an influenza virus inhibitor.

The subject of the present invention is the use of compounds 1.1.3-1.14.3 and 1.1.4-1.14.4 or their stereoisomers, pharmaceutically acceptable salts, solvates, hydrates and their crystalline or polycrystalline forms as pro-inhibitors (prodrugs) of the influenza virus.

The subject of the present invention is the use of inhibitors 1.1.2-1.14.2 or prodrugs 1.1.3-1.14.3 and 1.1.4-1.14.4 or their stereoisomers, pharmaceutically acceptable salts, solvates, hydrates and crystalline or polycrystalline forms thereof for the treatment or prevention of viral diseases.

The subject of this invention is an antiviral pharmaceutical composition containing a compound selected from the series 1.1.2-1.14.2 or the series of prodrugs 1.1.3-1.14.3 and 1.1.4-1.14.4 or a series of their stereoisomers, pharmaceutically acceptable salts, solvates, hydrates and their crystalline or polycrystalline forms in a therapeutically effective amount for the treatment or prevention of viral diseases.

More preferred is an antiviral pharmaceutical composition containing a compound selected from the series 1.1.2-1.14.2 or the series of prodrugs 1.1.3-1.14.3 and 1.1.4-1.14.4 or a series of stereoisomers thereof, pharmaceutically acceptable salts, solvates, hydrates and crystalline or polycrystalline forms thereof in a therapeutically effective amount for the treatment or prevention of influenza.

The subject of this invention is a method for the treatment and/or prevention of a viral disease, characterized by administering to the patient a compound selected from the series 1.1.2-1.14.2, or a series of prodrugs 1.1.3-1.14.3 and 1.1.4-1.14.4, or a series their stereoisomers, pharmaceutically acceptable salts, solvates, hydrates and their crystalline or polycrystalline forms or the pharmaceutical composition of this invention.

For the purpose of treating the above diseases, including influenza, in humans, the compounds of the present invention can be administered orally in the form of a powder, granule, tablet, capsule, pill, liquid and the like. or parenterally in the form of injection, suppository, transdermal drug, inhaler, etc. Effective doses of the present compounds can be mixed with excipients suitable for the dosage form, such as fillers, binders, humectants, disintegrants and lubricants, as necessary, to prepare pharmaceutical preparations. To prepare the injection, sterilization is carried out with a suitable carrier.

Pharmaceutical compositions in accordance with the present invention can be administered orally or parenterally. For oral administration, commonly used dosage forms such as tablets, granules, powder and capsules can be prepared according to conventional methods. For parenteral administration, a suitable conventional dosage form, such as an injection, can be used.

Effective doses of the compounds of the present invention can be mixed with various pharmaceutical excipients suitable for the dosage form, such as fillers, binders, disintegrants and lubricants, as necessary, to obtain pharmaceutical compositions.

The dose of the compounds of the present invention depends on the disease state, route of administration, age or weight of the patient. The usual oral dose for adults is 0.1 to 100 mg/kg per day, preferably 1 to 20 mg/kg per day. The dosage of the pharmaceutical composition of the present invention is preferably determined based on the age and weight of the patient, the type and severity of the disease, the route of administration, and the like. The usual oral dosage for adults is in the range of 0.05 to 100 mg/kg per day, preferably 0.1 to 10 mg/kg per day. The parenteral dose for adults varies considerably depending on the route of administration, but is usually in the range of 0.005 to 10 mg/kg per day, preferably 0.01 to 1 mg/kg per day. The dose may be administered once daily or may be divided into several daily doses. The compound of the present invention can be used in combination with other drugs (hereinafter referred to as combination drugs) to increase the activity of the compound, reduce the dose of the compound, or the like.

In the case of influenza treatment, the compound of the present invention can be used in combination with a neuraminidase inhibitor (eg, Oseltamivir, Zanamivir, Peramivir, AV-5080, Inabiru, etc.); RNA-dependent RNA polymerase inhibitor (eg, Favipiravir); M2 protein inhibitor (for example, Amantadine); PB2 protein binding inhibitor (cap-binding protein PB2, for example, VX-787); antiHA antibody (eg MNAA4549A); interferons (for example, Grippferon), interferon inducers (for example, Kagocel). Immune agonists (eg Nitazoxanide) are also possible. In this case, the administration time for the compound of the present invention and the combination drug is not limited. They can be given to patients to be treated at the same time or at different times. In addition, the compound of the present invention and the combination drug can be administered as two or more compositions, independent of each active ingredient or one composition containing each active ingredient.

The dose for combination drugs can be selected accordingly in relation to the clinical dose. The compounding ratio of the compounds of the present invention and the co-administered drugs can be suitably selected depending on the treatment to be treated, the route of administration, the disease to be treated, symptoms, drug combination and the like. For administration in humans, for example, 1 part by weight of the compounds of the present invention can be used in combination with 0.01-100 wt. including co-administered drugs.

The invention is illustrated by the following drawings.

In fig. Figure 1 shows fragments of the packaging of solvates 1.2.1-C ₂ H ₅ OH (A) and 1.2.1 i-C ₃ H ₇ OH (B), obtained as a result of X-ray diffraction analysis of crystals using the Platon program.

In fig. Figure 2 shows a general view of one of two symmetrically independent molecules of solvates 1.2.1-C ₂ H ₅ OH (A) and 1.2.1-iC ₃ H ₇ OH (B) in the representation of atoms by ellipsoids of thermal vibrations p = 50%. The solvate molecules of ethanol and isopropanol are not shown for clarity.

In fig. 3 - general view of molecule 1.9.1 in the representation of atoms by ellipsoids of thermal vibrations p = 50% (A) and the version obtained using the Platon program (B).

Best Mode for Carrying Out the Invention

The present invention is illustrated in more detail, but is not limited, by examples of the synthesis of compounds of general formula 1 and their testing.

- 20 044023

General procedures in chemistry. All chemicals and solvents were used as received from suppliers without further purification. The crude reaction mixtures were concentrated under reduced pressure by removing organic solvents on a rotary evaporator.

Nuclear magnetic resonance (NMR) spectra were recorded using a Bruker DPX-400 spectrometer at room temperature (RT) with tetramethylsilane as an internal standard. Chemical shifts (δ) are presented in parts per million (ppm), and signals are presented as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), or br s. (wide singlet).

High-resolution mass spectra (HRMS) were acquired on an Orbitrap Elite mass spectrometer (Thermo, Bremen, Germany) equipped with a HESI ion source.

High performance liquid chromatography (HPLC). The purity of the final compounds was determined by HPLC and was greater than 98%. HPLC conditions for purity assessment were as follows: Shimadzu HPLC, XBridge C18, 4.6 x 250 mm (3.5 µm); gradient of 0.1% TFA in 5% acetonitrile/water (A) and 0.1% TFA acetonitrile (B); flow rate 0.5 ml/min; collection time 20 min; wavelength, UV 214 and 254 nm. The preparative HPLC system included two sets of Shimadzu LC-8A pumps, a Shimadzu SCL 10Avp controller, and a Shimadzu SPD 10Avp detector. A Reprosil-Pur C18-AQ 10 µm, 250x20 mm column was used. The mobile phase had a gradient of 0.1% TFA in water (A) and 0.1% TFA in acetonitrile (B). LC/MS (LC/MS) was performed on a Sciex API 165 PE system using electrospray in positive ion mode [M+H]+ and a Shimadzu HPLC system equipped with a Waters XBridge C18 3.5 μm (4.6 x 150 mm) column. Diastereoisomers were separated on a chiral HPLC Phenomenex Lux 5u Cellulose-4, AXIA F, 250x30.00 mm. Flow rate: 25 ml/min. Detector: UV, 215 nm.

X-ray diffraction study of the samples was carried out on a Bruker APEX2 DUO diffractometer using CuKa radiation (graphite monochromator, ω-scanning). The structures were solved by the direct method and refined by least squares methods in the anisotropic full-matrix approximation using F ² _hk i. The hydrogen atoms of the OH groups of the solvate molecules of ethanol and isopropanol are localized in difference Fourier syntheses, and the positions of the remaining atoms are calculated geometrically. All hydrogen atoms are refined in the isotropic approximation using the riding model. Calculations were carried out using the SHELXTL PLUS [Sheldrick, GMYu Acta Cryst., 2008, A64:112-122] and Olex2 programs [Dolomanov, OV et al., J. Appl. Cryst., 2009, 42, 339-341].

Example 1. 6,7-Difluorothieno[3,2-c][2]benzothiepine-10(5H)-one (4.1) and 6,7-difluoro-5,10dihydrothieno[3,2-c][2]benzothiepine -10-ol (3) is obtained according to scheme 1.

To a solution of 30 g (174.3 mmol, 1 eq.) of 3,4-difluoro-2-methylbenzoic acid (3.1) in 300 ml of methanol, 207 g (1.74 mol, 10 eq.) of thionyl chloride were added dropwise. The mixture was heated to boiling under reflux and kept for 17 hours. The reaction mass was evaporated on a rotary evaporator, the residue was filled with water, the product was filtered and dried in air. 26.4 g of 3,4-difluoro-2-methylbenzoic acid methyl ester (3.2) were obtained in the form of a white crystalline powder.

1H NMR (400 MHz, CDCl ₃ ) δ 7.77-7.67 (m, 1H), 7.04 (dd, J=16.9, 8.9 Hz, 1H), 3.91 (s, 3H), 2.56 (d, J=2.7 Hz, 3H ).

To a solution of 28.0 g (150.4 mmol, 1 eq.) of the resulting ester 3.2 in 650 ml of carbon tetrachloride, 67.0 g (376.0 mmol, 2.5 eq.) of N-bromosuccinimide was added with stirring at room temperature. The reaction mass was heated to boiling and 360 mg (1.5 mmol, 0.01 eq.) of benzoyl peroxide was added in one portion. The reaction mass was stirred at reflux for 17 hours. The reaction mass was cooled to room temperature, the precipitate was filtered and washed with carbon tetrachloride. The filtrate was evaporated on a rotary evaporator, and the residue was purified by column chromatography (silica gel, ethyl acetate:hexane 1:9). 39.9 g of 2-(bromomethyl)-3,4-difluorobenzoic acid methyl ester (3.3) were obtained as a yellow oil, which crystallized on standing to give white crystals.

1H NMR (400 MHz, CDCl·,) δ 7.87-7.77 (m, 1H), 7.19 (dd, J=16.8, 8.8 Hz, 1H), 5.02 (d, J=2.1 Hz, 2H), 3.96 (s, 3H).

To a suspension of 12.5 g (90.3 mmol, 1.5 eq.) K ₂ CO ₃ and 3.9 g (12.0 mmol, 0.2 eq.) Cs ₂ CO ₃ in 70 ml of DMF was added 7.0 g (60.2 mmol, 1.1 eq.) thiophene-3- thiol (3.4) and the mixture was stirred at room temperature for 30 minutes. 14.5 g (54.7 mmol, 1 eq.) of 2-(bromomethyl)-3,4-difluorobenzoic acid methyl ester (3.3) was added to the reaction mass and the mixture was stirred at room temperature for 17 hours. The mixture was evaporated to dryness on a rotary evaporator, to 150 ml of ethyl acetate and 250 ml of water were added to the residue. The organic layer was separated and water was extracted with 150 ml of ethyl acetate. The combined organic extracts were washed with water, brine, dried over Na ₂ SO ₄ and evaporated to dryness on a rotary evaporator. The product was purified by column chromatography (silica gel, hexane: ethyl acetate = 1:0100:1-50:1-10:1). 12.2 g of 3,4-difluoro-2-[(3-thienylthio)methyl]benzoic acid methyl ester (3.5) were obtained as a yellow oil.

1H NMR (400 MHz, DMSO) δ 7.72-7.66 (m, 1H), 7.56 (dd, J=5.0, 3.0 Hz, 1H), 7.48-7.39 (m, 1H), 7.36 (dd, J=3.0, 1.2 Hz, 1H), 6.94 (dd, J=5.0, 1.2 Hz, 1H), 4.43 (d, J=1.6 Hz, 2H), 3.78 (s, 3H).

- 21 044023

To a solution of 7.9 g (26.3 mmol, 1 eq.) of 3,4-difluoro-2-[(3thienylthio)methyl]benzoic acid methyl ester (3.5) in 80 ml of methanol was added a solution of 5.0 g (118.4 mmol, 4.5 eq.) hydrate lithium hydroxide in 40 ml water. The mixture was stirred at room temperature for 17 hours and evaporated to dryness on a rotary evaporator. 100 ml of water and 2 N HCl were added to the residue to pH ~ 1-3 and the suspension was stirred at room temperature for 30 minutes. The precipitate is filtered and air dried. 7 g of 3,4-difluoro-2-[(3-thienylthio)methyl]benzoic acid (3.6) were obtained in the form of a white powder.

¹ H NMR (400 MHz, DMSO) δ 13.36 (br.s, 1H), 7.78-7.71 (m, 1H), 7.56 (dd, J=5.0, 3.0 Hz, 1H), 7.47-7.34 (m, 2H) , 6.98-6.92 (m, 1H), 4.48 (s, 2H).

To a solution of 1.0 g (3.5 mmol, 1 eq.) 3,4-difluoro-2-[(3-thienylthio)methyl]benzoic acid (3.6) in 120 ml of benzene was added 0.87 g (4.2 mmol, 1.2 eq.) PCl ₅ and the mixture was stirred and refluxed for 10 minutes, then cooled to room temperature. Argon (low current) was passed through the reaction mass for 10 minutes to remove hydrogen chloride. The reaction mass was cooled to 0°C and 0.91 g (3.5 mmol, 1 eq.) of tin tetrachloride was added dropwise. The reaction mass was stirred and refluxed for 10 minutes, then cooled. 70 ml of ether was added to the reaction mass and the mixture was washed twice with 50 ml of 2N HCl, 50 ml of water, and brine, dried over Na ₂ SO ₄ and evaporated to dryness on a rotary evaporator. The product was isolated by column chromatography (silica gel, hexane: ethyl acetate=30:1-20:1). We obtained 3.9 g of 6,7-difluorothieno[3,2-c][2]benzothipin10(5H)-one (3.7) in the form of a yellow powder.

1H NMR (400 MHz, DMSO) δ 8.07 (d, J=5.3 Hz, 1H), 7.64-7.47 (m, 2H), 7.23 (d, J=5.2 Hz, 1H), 4.39 (s, 2H).

0.28 g (7.3 mmol, 0.5 eq.) sodium borohydride and the reaction mass were stirred at room temperature for 30 minutes. The reaction mass was evaporated on a rotary evaporator, and 100 ml of sat. was added to the residue. solution of NaHCO ₃ in water. The product was filtered, washed with water and air dried. 3.9 g of 6,7-difluoro-5,10-dihydrothieno[3,2-c][2]benzothipin-10-ol (3) were obtained in the form of a yellowish powder.

1H NMR (400 MHz, DMSO) δ 7.47-7.26 (m, 3H), 6.84-6.63 (m, 2H), 6.27 (s, 1H), 4.59 (d, J=14.2 Hz, 1H), 4.42 (d, J=14.3 Hz, 1H).

Example 2. 7,8-Difluoro-4,9-dihydrothieno[2,3-c][2]benzothipin-4-ol (4) was prepared according to scheme 2.

To a suspension of 8.9 g (64.5 mmol) of K2CO3 and 2.8 g (8.6 mmol) of _Cs2CO3 in 50 ml of DMF, 5.0 g (43.0 mmol) of thiophene-2-thiol (4.1 ₎ was added and the mixture was stirred at room temperature for 30 min. 11.4 g (43.0 mmol) of 2-(bromomethyl)-3,4difluorobenzoic acid methyl ester (3.3) was added to the reaction mass and the mixture was stirred at room temperature for 17 hours. The resulting mixture was evaporated to dryness on a rotary evaporator, and 150 ml of ethyl acetate was added to the residue and 250 ml of water. The organic layer was separated and the aqueous layer was extracted with 150 ml of ethyl acetate. The combined organic extracts were washed with water, brine, dried over Na ₂ SO ₄ and evaporated to dryness on a rotary evaporator. The product was purified by column chromatography (silica gel, dichloromethane hexane 1:4). 9.2 g of 3,4-difluoro-2-[(2-thienylthio)methyl]benzoic acid methyl ester (4.2) were obtained as a yellow oil.

1H NMR (400 MHz, DMSO) δ 7.76-7.70 (m, 1H), 7.64 (dd, J=5.3, 1.2 Hz, 1H), 7.47 (dd, J=18.1, 8.6 Hz, 1H), 7.00 (dd, J =5.3, 3.6 Hz, 1H), 6.97-6.93 (m, 1H), 4.41 (s, 2H), 3.77 (s, 3H).

A solution of 2.6 g (45.9 mmol) KOH in 50 ml of water was added to a solution of 9.2 g (30.6 mmol) of 3,4-difluoro-2-[(2-thienylthio)methyl]-benzoic acid methyl ester (4.2) in 50 ml of methanol. The mixture was stirred at room temperature for 17 hours, then the methanol was distilled off under reduced pressure. The aqueous residue was extracted twice with 50 ml of ether and then acidified with 2 N HCl to pH ~ 1-3. The precipitate that formed was stirred at room temperature for 30 minutes. The precipitate was filtered off and dried in air. 8.4 g of 3,4-difluoro-2-[(2-thienylthio)methyl]benzoic acid (4.3) was obtained in the form of a white powder.

1H NMR (400 MHz, DMSO) δ 13.36 (s, 1H), 7.81-7.74 (m, 1H), 7.63 (dd, J=5.2, 1.2 Hz, 1H), 7.43 (dd, J=17.8, 8.7 Hz, 1H), 7.03-6.94 (m, 2H), 4.44 (s, 2H).

To a solution of 4.2 g (4.7 mmol) of 3,4-difluoro-2-[(2-thienylthio)methyl]benzoic acid (4.3) in 70 ml of benzene, 3.7 g (17.6 mmol) of PCl ₅ was added and the mixture was stirred and refluxed for 10 minutes, then cooled to room temperature. Argon (low current) was passed through the reaction mixture for 10 minutes to remove hydrogen chloride. The reaction mass was cooled in an ice-water bath to 0°C and 3.8 g (14.7 mmol, 1 eq.) of tin tetrachloride was added dropwise. The reaction mass was stirred and refluxed for 10 minutes, then cooled to room temperature. 70 ml of ether was added to the reaction mass and washed twice with 50 ml of 2 N HCl, 50 ml of water, brine, dried over Na ₂ SO ₄ and evaporated to dryness on a rotary evaporator. The product was isolated by column chromatography (silica gel, hexane:ethyl acetate 20:1). 1.8 g of 7,8-difluorothieno[2,3-c][2]benzothipin-4(9H)-one (4.4) was obtained in the form of a yellow powder.

1H NMR (400 MHz, DMSO) δ 7.62-7.45 (m, 4H), 4.49 (s, 2H).

- 22 044023

To a suspension of 1.8 g (6.7 mmol) 7,8-difluorothieno[2,3-с][2]benzothipin-4(9Н)-one (4.4) in 50 ml of methanol was added 0.25 g (6.7 mmol) sodium borohydride and the reaction mass stirred at room temperature for 30 minutes. The reaction mass was evaporated on a rotary evaporator, the residue was suspended in 50 ml of methylene chloride, and washed with 50 ml of a saturated NaHCO ₃ solution. The methylene solution was dried over Na ₂ SO ₄ and evaporated to dryness. The residue in the flask crystallized. The product was washed with water and air dried. 1.7 g of 7,8-difluoro-4,9-dihydrothieno[2,3-c][2]benzothipin-4-ol (4) was obtained in the form of a yellowish powder.

¹ H NMR (400 MHz, DMSO) δ 7.35-7.25 (m, 2H), 7.25-7.18 (m, 1H), 7.07 (d, J=4.7 Hz, 1H), 6.27 (d, J=4.0 Hz, 1H ), 5.96 (d, J=3.9 Hz, 1H).

Example 3. (3,4-Difluorophenyl)(phenyl)methanol (5) is obtained similarly to the synthesis of alcohols 3 and 4 from the corresponding ketones 3.7 and 4.4 (3,4-difluorophenyl)(phenyl)methanol (5).

LC-MS (ESI, 20 min), 221 (M+H)+.

¹ H NMR (DMSO-d ₆ , 400 MHz) δ 7.29-7.42 (m, 6H), 7.17-7.24 (m, 2H), 6.06 (d, J=4.0 Hz, 1H), 5.71 (d, J=4.0 Hz, 1H).

Example 4. (3,4-Difluorophenyl)(2-methylsulfanylphenyl)methanol (6).

To a suspension of 11.0 g (79.4 mmol, 1.5 eq.) K2CO3 and 3.5 g (10.6 mmol, 0.2 eq.) Cs ₂ CO ₃ in 50 ml of DMF was added 10.0 g (52.9 mmol, 1 eq.) 1-bromo-2-thiophenol (6.1) and the reaction mass was stirred at room temperature for 30 minutes. 11.3 g (79.4 mmol, 1.5 eq.) of sweeping were added to the reaction mixture and the mixture was stirred at room temperature for 1 hour. The mixture was poured into 200 ml of water, extracted with diethyl ether, the combined organic phases were washed with water, brine and dried over Na ₂ SO ₄ . The solvent was evaporated to dryness on a rotary evaporator and the product was used in the next step without further purification. 10.4 g (97%) of 1-bromo-2methylsulfanylbenzene (6.2) was obtained as a yellow oil.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.54 (dd, J=7.9, 1.2 Hz, 1H), 7.36-7.28 (m, 1H), 7.15 (d, J=7.9 Hz, 1H), 7.02 (td, J=7.8, 1.4 Hz, 1H), 2.49 (s, 3H).

To a solution of 18.7 g (118.3 mmol, 1 eq.) of 3,4-difluorobenzoic acid (6.4) in 374 ml of methylene chloride, 20.1 g (124.2 mmol, 1.05 eq.) CDI was slowly added and stirred at room temperature for 15 minutes until the CO2 release. 12.7 g (130.1 mmol, 1.1 eq.) of N,Ο-dimethylhydroxylamine hydrochloride was added to the reaction mixture and stirred at room temperature for 17 hours. The mixture was diluted with 400 ml of water, the layers were separated, and the aqueous layer was extracted with methylene chloride (twice 100 ml each). The combined organic layers are washed with 200 ml of water, 100 ml of brine, dried over Na ₂ SO ₄ and rotary evaporated. 12 g (50%) of 3,4-difluoro-M-methoxy-M-methylbenzamide (6.5) are obtained as a colorless oil.

¹ N NMR (400 MHz, CDCl·,) δ 7.66-7.57 (m, 1H), 7.57-7.49 (m, 1H), 7.20 (dd, J=18.1, 8.3 Hz, 1H), 3.56 (s, 3H) , 3.37 (s, 3H).

To a suspension of 1.49 g (61.4 mmol, 1.2 eq.) of Mg in 60 ml of THF, 10.4 g (51.2 mmol, 1 eq.) of 1-bromo-2-(methylsulfanyl)benzene (6.2) was added and the mixture was stirred at room temperature for 2 hours. A solution of [2-(methylsulfanyl)phenyl]magnesium bromide (6.3) in THF was obtained.

A solution of [2 -(methylsulfanyl)phenyl]magnesium bromide (6.3) in THF, maintaining the temperature (-78)-(-60)°C. The mixture was stirred at (-70)°C for 15 minutes, then warmed to room temperature and stirred for 17 hours. The reaction mass was cooled to 0°C, 200 ml of 1 N HCl was added to the reaction mass and the mixture was stirred in an ice bath in for 15 minutes. 50 ml of ethyl acetate and 100 ml of water were added to the mixture, the organic layer was separated and the aqueous layer was extracted with ethyl acetate (twice 100 ml), the combined organic extract was washed with water (twice 50 ml), brine, dried over Na ₂ SO ₄ and evaporated on a rotary evaporator dry. The product was purified by column chromatography (silica gel, ethyl acetate:hexane=30:1-20:1). 3.6 g of (3,4-difluorophenyl)(2methylsulfanylphenyl)methanone (6.6) was obtained as a colorless oil.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.71-7.64 (m, 1H), 7.58-7.47 (m, 2H), 7.46-7.41 (m, 1H), 7.39-7.34 (m, 1H), 7.28-7.22 (m, 2H), 2.44 (s, 3H).

To a solution of 3.6 g (13.6 mmol, 1 eq.) of (3,4-difluorophenyl)(2-methylsulfanylphenyl)methanone (6.6) in 36 ml of methanol was added 0.26 g (6.8 mmol, 0.5 eq.) NaBH ₄ and the reaction mass stirred at room temperature for 30 minutes. The reaction mass was evaporated on a rotary evaporator, 10 ml of 2 N HCl was added to the residue, and the mixture was stirred at room temperature for 5 min. A saturated solution of 100 ml of NaHCO3 was added to the mixture in portions and the organic product was extracted with ethyl acetate (3 times 60 ml). The combined extract was washed with brine, dried over Na ₂ SO ₄ and evaporated to dryness on a rotary evaporator. The product was purified by column chromatography (silica gel, hexane:ethyl acetate=30:1-15:1-9:1-7:1-5:1). 2.9 g (77%) of (3,4-difluorophenyl)(2-methylsulfanylphenyl)methanol (6) was obtained as a colorless oil.

¹ H NMR (400 MHz, DMSO) δ 7.51 (d, J=7.5 Hz, 1H), 7.38-7.26 (m, 4H), 7.23-7.17 (m, 1H), 7.13-7.07

- 23 044023 (m, 1H), 6.10 (d, J=4.3 Hz, 1H), 5.97 (d, J =4.3 Hz, 1H), 2.44 (s, 3H).

Example 5. (12aR)-7-(Benzyloxy)-12-(6,7-difluoro-5,10-dihydro[3,2-c][2]benzothipin-10-yl)3,4,12,12a -tetrahydro-1H-[1,4]oxazino[3,4-c1pyrido[2,1-e][1,2,4]triazine-6,8-diones 1.1.1 and its diastereomers 1.2.1, 1.3. 1.

To a mixture of 3.3 g (10.1 mmol, 1 eq.) 7-(benzyloxy)-3,4,12,12a-tetrahydro-1H-[1,4]oxazino[3,4c]pyrido[2,1-f][ 1,2,4]triazine-6,8-dione (2) and 4.1 g (15.2 mmol, 1.5 eq.) 6,7-difluoro-5,10dihydrothieno[3,2-c][2]benzothiepine-10- ol (3) was added 64.3 g (101 mmol, 10 eq.) 50% T ₃ P in ethyl acetate. The reaction mass was stirred at room temperature for 5 days, 200 ml of sat. was poured into the mixture. NaHCO ₃ solution and 200 ml of ethyl acetate. The aqueous solution was separated and the ethyl acetate layer was again washed with sat. NaHCO ₃ solution, brine, dried over Na ₂ SO ₄ and evaporated on a rotary evaporator. The product was purified by column chromatography (EtOAc). Product 1.1.1 was obtained as a white powder (5.7 g, 97%). The resulting mixture of diastereomers was recrystallized from 170 ml of ethyl acetate. The precipitate was filtered and washed on the filter with ethyl acetate. Diastereomer 1.3.1 was obtained in the form of a white powder 1.46 g (25%). The filtrate was evaporated to dryness on a rotary evaporator, the residue was separated on chiral HPLC, and diastereomer 1.2.1 was obtained, which came out of the column first, and diastereomer 1.3.1, which came out of the column second. Additionally, 2.8 g of diastereomer 1.2.1 was recrystallized from 100 ml of isopropanol. The absolute configuration of diastereomers 1.2.1 and 1.3.1 was established on the basis of X-ray diffraction data (Table 4) of the orthorhombic solvates (Figs. 1 and 2) 1.2.1-C2H5OH and 1.2.1 i-C ₃ H ₇ OH (ratios 4: 3 and 8:3, respectively) obtained by crystallization of diastereomer 1.2.1 from ethanol and isopropanol.

Table 4

Basic crystallographic data and refinement parameters of 1.2.1-C2H5OH and 1.2.1-1-C ₃ H ₇ OH

Options 1.2.1'C ₂ H ₅ OH 1.2.1-/-СзНтОН Gross formula CmHnoFsNuOisSs C241H2G8F16N24O35 S16 Molecular mass 2456.69 4817.26 T,K 120 120 Crystalline system Rhombic Rhombic Spatial group Ρ2ι2ι2 P21212 Z 2 1 a, a 15.2405(4) 15.2647(3) b, a 33.4723(8) 33.3600(7) s, A 11.2776(3) 11.4164(3) a, ° 90.00 90.00 β,° 90.00 90.00 γ,° 90.00 90.00 v, A ³ 5753.1(3) 5813.6(2) yvych, ghsm' ³ 1.418 1.376 μ, cm' ¹ 21.77 21.35 F(000) 2556 2502 20shaKh, ° 135 135 Number of measured reflections 76014 82664 Number of independent reflections 10279 10421 Number of reflections with Ι>2σ(Ι) 9918 9852

- 24 044023

Number of parameters to be specified 779 760 R.I. 0.0347 0.0522 wR2 0.0904 0.1513 G.O.F. 1.077 1.107 Residual electron density, e ^x A dmax/dmin) 0.692/-0.413 0.810/-0.390

Diastereomer 1.2.1:

LC MS, m/z 580 (M+1);

1H NMR (400 MHz, DMSO) δ 7.76 (d, J=5.3 Hz, 1H), 7.56 (d, J=7.4 Hz, 2H), 7.40-7.29 (m, 3H), 7.21-7.11 (m, 1H) , 7.03 (d, J=7.7 Hz, 1H), 6.91 (d, J=5.4 Hz, 1H), 6.83-6.75 (m, 1H), 5.76-5.65 (m, 2H), 5.34-5.22 (m, 2H ), 5.15 (d, J=11.0 Hz, 1H), 4.56-4.48 (m, 2H), 4.13 (d, J=14.4 Hz, 1H), 3.99-3.92 (m, 1H), 3.73-3.66 (m, 1H), 3.39 (t, J=10.3 Hz, 1H), 2.96-2.86 (m, 1H), 1.04 (d, J=6.0 Hz, 1H).

Diastereomer 1.3.1:

LC MS, m/z 580 (M+1);

¹ H NMR (400 MHz, DMSO) δ 7.57 (d, J=7.0 Hz, 2H), 7.51-7.29 (m, 6H), 7.23 (d, J=7.7 Hz, 1H), 6.77 (d, J=5.4 Hz, 1H), 5.88-5.80 (m, 2H), 5.49-5.34 (m, 1H), 5.25 (d, J=10.7 Hz, 1H)3 5.14 (d, J=10.8 Hz, 1H), 4.50-4.40 (m, 1H), 4.35 (dd, J=9.9, 2.9 Hz, 1H), 4.07 (d, J=9.8 Hz, 1H), 3.85 (dd, J=10.7, 2.8 Hz, 1H), 3.70-3.59 ( m, 1H), 3.36 (t, J=10.3 Hz, 1H), 3.28-3.19 (m, 1H), 2.99-2.81 (m, 1H).

Example 6. (12aR)-7-hydroxy-12-(6,7-difluoro-5,10-dihydro[3.2-c][2]benzothipin-10-yl)-3,4,12,12atetrahydro-1H- [1,4]oxazino[3,4-c]pyrido[2,1-e][1,2.4]triazine-6,8-dione 1.1.2 and its diastereomers 1.2.2 and 1.3.2.

To a solution of 0.173 mol (1 eq.) (12aR)-7-(benzyloxy)-12-(6,7-difluoro-5,10-dihydro[3,2c][2]benzothipin-10-yl)-3, 4,12,12a-tetrahydro-1H-[1,4]oxazino[3,4-c]pyrido[2,1-e][1,2,4]triazine-6,8cyone (1.1.1) or its diastereomers 1.2.1 and 1.3.1 in 15 ml of dimethylacetamide, 36 mg (0.863 mmol, 5 eq.) LiCl was added, the reaction mass was heated to 80°C and stirred for 2 hours. The reaction mass was evaporated to dryness on a rotary evaporator, and the residue was added 50 ml of aqueous 0.5N HCl and the product was extracted with ethyl acetate (three times 30 ml). The combined extract was washed with brine, dried over Na ₂ SO ₄ and evaporated to dryness on a rotary evaporator. The residue was purified by HPLC and, respectively, inhibitor 1.1.2 (LC MS, m/z 454 (M+1)) or its diastereomers 1.1.2, 1.3.2 (LC MS, m/z 454 (M+1)) were obtained. ). Inhibitor 1.1.2 is, according to NMR data, a mixture of diastereomers 1.2.2 and 1.3.2 in a ratio of 65:35.

Diastereomer 1.2.2:

LC MS, m/z 454 (M+1);

1H NMR (400 MHz, DMSO) δ 7.53-7.37 (m, 3H), 7.14 (d, J=7.8 Hz, 1H), 6.77 (d, J=5.3 Hz, 1H), 5.86 (s, 1H), 5.69 (d, J=7.7 Hz, 1H), 5.41 (d, J=14.1 Hz, 1H), 4.42 (d, J=10.6 Hz, 2H), 4.09 (d, J=14.2 Hz, 1H), 3.90-3.81 (m, 1H), 3.70-3.56 (m, 2H), 3.46-3.39 (m, 2H), 3.00 (s, 1H).

Diastereomer 1.3.2:

LC MS, m/z 454 (M+1);

1H NMR (400 MHz, DMSO) δ 7.76 (d, J =5.3 Hz, 1H), 7.20 (dd, J=18.5, 8.3 Hz, 1H), 7.05-6.87 (m, 3H), 5.84 (s, 1H) , 5.56 (d, J=7.7 Hz, 1H), 5.28 (d, J=14.9 Hz, 1H), 4.57 (dd, J=9.8, 2.8 Hz, 1H), 4.49 (d, J=11.8 Hz, 1H) , 4.13 (d, J=14.5 Hz, 1H), 4.01-3.93 (m, 1H), 3.76-3.68 (m, 1H), 3.62 (t, J=10.3 Hz, 1H), 3.49-3.39 (m, 2H ), 3.05-2.94 (m, 1H).

Example 7 12,12a-hexahydro-1H-[1,4]oxazino[3,4-c]pyrido[2,1-f ^: ][1,2.4]triazin-6-yl}oxy)methyl methyl carbonate 1.1.4 and its diastereoisomers are 1.2.4, 1.3.4.

To a suspension of 0.5 mmol of compound 1.1,2 or its diastereomer 1.2.2, 1.3.2 in 1 ml of dimethylacetamide, 93 mg (0.75 mmol) of chloromethyl methyl carbonate, 137 mg (1.0 mmol) of potassium carbonate and 82 mg (0.5 mol) of potassium iodide were added , then the reaction mass was heated to 60°C and stirred for 48 hours. The reaction mass was evaporated to dryness on a rotary evaporator, 10 ml of 0.5 N HCl was added to the residue, and the product was extracted with EtOAc (30 ml each). The combined extract was washed with 30 ml of saturated aqueous NaHCO ₃ solution, dried over Na ₂ SO ₄ and evaporated to dryness on a rotary evaporator. The residue was purified by HPLC. We respectively obtained prodrug 1.1.4 (LC MS, m/z 578 (M+1)) or its diastereoisomers 1.2.4 (LC MS, m/z 578 (M+1)), 1.3.4 (LC MS, m/ z 578 (M+1)).

Example 8 12,12a-hexahydro-1H-[1,4]oxazino[3,4-c1dirido[2,1-f][1,2,4]triazin-6-yl methyl carbonate 1.1.3 and its diastereoisomers 1.2.3 , 1.3.3.

Prodrugs 1.1.3 (LC MS, m/z 548 (M+1), 1.2.3 (LC MS, m/z 548 (M+1)) and 1.3.3 (LC MS, m/z 548

- 25 044023 (M+1)) were prepared analogously to example 6, using methyl chloroformic acid for the carbomethoxylation of compounds 1.1.2, 1.2.2 and 1.3.2, respectively.

Example 9 -1H-[1.4]oxazino[3,4-c]pyrido[2,1-G][1,2,4]triazine-6.8-diones 1.4.1 and its diastereomers 1.5.1, 1.6.1.

0.52 g (4.6 mmol) of mesyl chloride and the resulting mixture was stirred at room temperature for 24 hours. Then 7-(benzyloxy)-3,4,12,12a-tetrahydro-1H-[1,4]oxazino[3,4c]pyrido[2,1-£] was added to the mixture [1,2,4]triazine-6,8-dione (2) and stirred at 70°C for another 24 hours. Pyridine was evaporated under reduced pressure, the residue was dissolved in 100 ml of methylene chloride, washed with 50 ml of water, dried over sodium sulfate , evaporated. The resulting product was purified by silica gel column chromatography with pure ethyl acetate. We obtained (12aR)-7-(benzyloxy)-12-(7,8-difluoro-4,9-dihydrothieno[2,3c][2]benzothipin-4-yl)-3,4,12,12a-tetrahydro- 1H-[1,4]oxazino[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8dione 1.4.1, consisting of diastereomers 1.5.1, 1.6.1, which were isolated on a preparative chiral column Phenomenex Lux 5u Cellulose-4, AXIA F, 250x30.00 mm. Flow rate: 25 ml/min. Detector: UV, 254 nm. The mobile phase has a constant composition of acetonitrile: isopropanol 80:20.

(12aR)-7-(Benzyloxy)-12-(7,8-difluoro-4,9-dihydrothieno[2,3-c][2]benzothipin-4-yl)-3,4,12,12atetrahydro-1H -[1,4]oxazino[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8-dione (1.4.1):

LC MS, m/z 580 (M+1);

¹ НNMR (400 MHz, DMSO) δ 7.61-7.51 (m, 2.6H), 7.48-7.29 (m, 3.8H), 7.25 (d, J=7.7 Hz, 0.4H), 7.20-7.09 (m, 1H) , 7.02 (dd, J=12.0, 6.5 Hz, 1.2H), 6.76-6.67 (m, 0.6H), 6.26 (d, J=5.2 Hz, 0.4H), 5.83 (s, 0.4H), 5.73-5.66 (m, 1.2H), 5.48 (d, J=14.6 Hz, 0.4H), 5.35-5.26 (m, 1.6H), 5.18-5.10 (m, 1H), 4.54-4.41 (m, 1H), 4.36- 4.29 (m, 1H), 4.24 (d, J=14.5 Hz, 0.6H), 4.18 (d, J=14.3 Hz, 0.4H), 3.94 (d, J=7.6 Hz, 0.6H), 3.84 (d, J=7.8 Hz, 0.4H), 3.72-3.61 (m, 1H), 3.42-3.32 (m, 1.4H), 3.29-3.20 (m, 1H), 2.91 (t, J=11.0 Hz, 0.4H), 2.83 (t, J=10.9 Hz, 0.6H).

Diastereomer 1.5.1:

LC MS, m/z 580 (M+1);

1H NMR (400 MHz, DMSO) δ 7.54 (t, J=5.5 Hz, 3H), 7.38 (t, J=7.2 Hz, 2H), 7.33 (t, J=7.2 Hz, 1H), 7.22-7.11 (m , 1H), 7.02 (dd, J=12.0, 6.5 Hz, 2H), 6.76-6.67 (m, 1H), 5.73-5.66 (m, 2H), 5.35-5.26 (m, 2H), 5.14 (d, J =10.9 Hz, 1H), 4.49 (d, J=11.8 Hz, 1H), 4.32 (d, J=6.9 Hz, 1H), 4.24 (d, J=14.5 Hz, 1H), 3.94 (d, J=7.6 Hz, 1H), 3.68 (d, J=8.8 Hz, 1H), 3.39-3.34 (m, 1H), 3.29-3.21 (m, 1H), 2.83 (t, J=10.9 Hz, 1H).

Diastereomer 1.6.1:

LC MS, m/z 580 (M+1);

1H NMR (400 MHz, DMSO) δ 7.57 (d, J=6.9 Hz, 2H), 7.48-7.29 (m, 5H), 7.25 (d, J=7.7 Hz, 1H), 7.11 (d, J=5.2 Hz , 1H), 6.26 (d, J=5.2 Hz, 1H), 5.83 (s, 1H), 5.48 (d, J=14.6 Hz, 1H), 5.29 (d, J=11.0 Hz, 1H), 5.12 (d , J=10.9 Hz, 1H), 4.46 (d, J=12.0 Hz, 1H), 4.33 (d, J=7.0 Hz, 1H), 4.18 (d, J=14.3 Hz, 1H), 3.84 (d, J =7.8 Hz, 1H), 3.65 (d, J=11.2 Hz, 1H), 3.42-3.34 (m, 2H), 3.25 (t, J=10.7 Hz, 1H), 2.91 (t, J=11.0 Hz, 1H ).

Example 10 -1H-[1,4]oxazino[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8-diones 1.4.2 and its diastereomers 1.5.2 and 1.6. 2.

To a solution of 0.14 mmol of 7-benzyloxy derivative 1.4.2, 1.5.2 or 1.6.2 in 5 ml of dimethylacetamide, 9 mg (0.70 mmol) LiCl was added, the reaction mass was heated to 80°C and stirred for 3 hours. The reaction mass was evaporated to rotary evaporator to dryness, and the residue was purified on HPLC. The corresponding product 1.4.2, 1.5.2 and 1.6.2 were obtained.

7-G hydroxy-(12aR)-12-(7,8-difluoro-4,9-dihydrothieno[2,3-c][2]benzothipin-4-yl)-3,4,12,12atetrahydro-1H- [1,4]oxazino[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8-diones 1.4.2:

LC MS, m/z 454 (M+1);

1H NMR (400 MHz, DMSO) δ 7.53 (d, J=5.2 Hz, 0.5H), 7.44 (dd, J=18.2, 8.4 Hz, 0.5H), 7.38-7.31 (m, 0.5H), 7.27-7.13 (m, 1.5H), 7.03 (d, J=5.3 Hz, 0.5H), 6.97 (d, J=7.7 Hz, 0.5H), 6.92-6.85 (m, 0.5H), 6.42 (d, J=5.3 Hz, 0.5H), 5.87 (s, 0.5H), 5.80 (s, 0.5H), 5.69 (d, J=7.6 Hz, 0.5H), 5.56 (d, J=7.7 Hz, 0.5H), 5.48 ( d, J=13.5 Hz, 0.5H), 5.33 (d, J=14.4 Hz, 0.5H), 4.53-4.35 (m, 2H), 4.30-4.14 (m, 1H), 3.96 (dd, J=10.6, 2.7 Hz, 0.5H), 3.90-3.81 (m, 0.5H), 3.69 (t, J=12.4 Hz, 1H), 3.59 (t, J=10.3 Hz, 1H), 3.46-3.41 (m, 1H), 3.08-2.97 (m, 0.5H), 2.91 (t, J=11.0 Hz, 0.5H).

Diastereomer 1.5.2:

LC MS, m/z 454 (M+1);

1H NMR (400 MHz, DMSO) δ 11.80 (br.s, 1H), 7.43 (dd, J=18.2, 8.6 Hz, 1H), 7.38-7.29 (m, 1H), 7.17 (t, J=6.3 Hz, 2H), 6.42 (d, J=5.2 Hz, 1H), 5.87 (s, 1H), 5.69 (d, J=7.6 Hz, 1H), 5.49 (d, J=14.7 Hz, 1H), 4.55-4.33 ( m, 2H), 4.19 (d, J=14.4 Hz, 1H), 3.86 (d, J=8.0 Hz, 1H), 3.67 (d, J=9.2 Hz, 1H), 3.59 (t, J=10.4 Hz, 1H), 3.40 (t, J=10.5 Hz, 1H), 3.03 (t, J=11.0 Hz, 1H).

Diastereomer 1.6.2:

LC MS, m/z 454 (M+1);

1H NMR (400 MHz, DMSO) δ 11.77 (s, 1H), 7.53 (d, J=5.1 Hz, 1H), 7.21 (dd, J=17.9, 8.6 Hz, 1H), 7.03

- 26 044023 (d, J=5.2 Hz, 1H), 6.97 (d, J=7.6 Hz, 1H), 6.93-6.82 (m, 1H), 5.80 (s, 1H), 5.56 (d, J=7.6 Hz , 1H), 5.33 (d,

J=14.6 Hz, 1H), 4.48 (d, J=12.6 Hz, 1H), 4.39 (d, J=7.6 Hz, 1H), 4.25 (d, J=14.5 Hz, 1H), 3.96 (d, J= 8.6 Hz,

1H), 3.71 (d, J=9.3 Hz, 1H), 3.59 (t, J=10.2 Hz, 1H), 3.42 (t, J=10.8 Hz, 1H), 2.91 (t, J=11.2 Hz, 1H) .

Example 11 4,6,8,12,12a-hexahydro-1H-[1,4]oxazino[3,4-c]pyrido[2,1-f][1,2,4]triazin-7-yl}oxy) methyl carbonates 1.5.4 and ({(12aR)-12-[(4R)-7,8-difluoro-4,9-dihydrothieno[2,3-c][2]benzothipin-4-yl]-6,8 -dioxo-3,4,6,8,12,12ahexahydro-1H-[1,4]oxazino[3,4-c]pyrido[2,1-f][1,2,4]triazin-7-yl }hydroxy)methyl carbonate 1.6.4.

Received under the conditions described in example 6, based on the corresponding diastereomers 1.5.2 and 1.6.2.

Prodrug 1.5.4:

LC MS, m/z 578 (M+1).

Prodrug 1.6.4:

LCMS, m/z 578 (M+1).

Example 12 -c]pyrido[2,1-G][1,2,4]triazine-6,8-dione 1.7.1 and its diastereomers 1.8.1, 1.9.1.

To a mixture of 900 mg (2.7 mmol) (12aR)-7-(benzyloxy)-3,4,12,12a-tetrahydro-1H-[1,4]oxazino[3,4c]pyrido[2,1-£][ 1,2,4]triazine-6,8-dione (2) and 605 mg (2.7 mmol) (3,4-difluorophenyl)(phenyl)methanol (5) in 17.5 g (27.0 mmol) 50% T ₃ R in 528 mg (5.4 mmol) of methanesulfonic acid were added to ethyl acetate and stirred at 70°C for 16 hours. The reaction mass was cooled to room temperature, 50 ml of ethyl acetate was added and washed with 50 ml of a saturated NaHCO ₃ solution, dried over Na2SO4 and evaporated to dryness on a rotary evaporator. The resulting product was purified by silica gel column chromatography with pure ethyl acetate. After evaporation of the corresponding fractions, 240 mg of product (1.7.1) was obtained: LC MS (20 min): Rt=15.53 (220 nm), m/z 530 (M+1), consisting of (12aR)-7-(benzyloxy) -12-[(R)-(3,4difluorophenyl)(phenyl)methyl]-3,4,12,12a-tetrahydro-1H-[1,4]oxazino[3,4-c]pyrido[2,1- f][1,2,4]triazine6,8-dione (1.8.1) and (12aR)-7-(benzyloxy)-12-[(S)-(3,4-difluorophenyl)(phenyl)methyl] - 3,4,12,12a-tetrahydro1H-[1,4]oxazino[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8-dione (1.9.1) , which were separated on a preparative chiral column Phenomenex Lux 5u Cellulose-4, AXIA F, 250x30.00 mm. Flow rate: 25 ml/min. Detector: UV, 254 nm. The mobile phase has a constant composition of acetonitrile-isopropanol 80:20.

Diastereomer 1.8.1:

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.61 (d, J=6.6 Hz, 2H), 7.40-7.33 (m, 4H), 7.25-7.17 (m, 3H), 7.12 (t, J=7.6 Hz, 2H), 6.93 (d, J=7.5 Hz, 2H), 6.73 (d, J=7.7 Hz, 1H), 5.75 (d, J=7.9 Hz, 1H), 5.59 (d, J=10.7 Hz, 1H) , 5.45 (d, J=10.8 Hz, 1H), 5.39 (s, 1H), 4.65 (d, J=13.8 Hz, 1H), 4.58-4.50 (m, 1H), 3.97 (d, J=10.4 Hz, 1H), 3.73 (d, J=8.9 Hz, 1H), 3.37-3.22 (m, 2H), 2.96 (t, J=10.9 Hz, 1H).

Diastereomer 1.9.1:

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.62 (d, J=7.2 Hz, 2H), 7.50-7.32 (m, 8H), 7.06-6.95 (m, 1H), 6.91-6.76 (m, 2H), 6.61-6.46 (m, 1H), 5.94 (d, J=7.9 Hz, 1H), 5.64 (d, J=10.7 Hz, 1H), 5.47 (d, J=10.8 Hz, 1H), 5.36 (s, 1H ), 4.60 (t, J=12.8 Hz, 2H), 3.95 (d, J =10.7 Hz, 1H), 3.71 (d, J=8.8 Hz, 1H), 3.39-3.20 (m, 2H), 2.99-2.83 (m, 1H).

After crystallization from a dichloromethane-hexane mixture of diastereomer 1.9.1, the resulting crystals were analyzed by X-ray diffraction. The data obtained (Fig. 3, Table 5) confirmed its stereochemical structure.

- 27 044023

Table 5

Basic crystallographic data and diastereomer refinement parameters 1.9.1

Gross formula C ₃ oH25F ₂ N ₃ 04 Molecular mass 529.53 T, K 120 Crystal system Triclinic Space group Pl Z 1 a, a 7.2293(7) b, a 9.5120(9) s, A 10.0046(9) a, ° 70.501(2) R/ 85.503(2) b° 87.171(2) V, A ³ 646.32(11) Yvyi, G*cm' ³ 1.360 μ, cm' ¹ 1.01 F(000) 276 2qmax, ° 56 Number of measured reflections 7474 Number of independent reflections 6090 Number of reflections with I>2s(I) 5042 Number of parameters to be specified 352 R1 0.0459 wR2 0.0938 G.O.F. 1.0188 Residual electron density, exA' ³ (dmax/dmin) 0.259-0.228

Example 13. (12aR)-7-G hydroxy-12-[(3,4-difluorophenyl)(pheni)methyl]-3,4,12,12a-tetrahydro-1H-

[1,4]oxazino[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8-dione (1.7.2) and its diastereomers 1.8.2 and 1.9.2 .

To a solution of 64 mg (0.12 mmol) (12aR)-7-(benzyloxy) derivative 1.7.1, 1.8.1 or 1.91 in 3 ml of dimethylacetamide, 40 mg (0.95 mmol) LiCl was added, the reaction mass was heated to 80°C and stirred for 3 hours. The reaction mass was evaporated to dryness on a rotary evaporator, the residue was purified on HPLC.

- 28 044023

We obtained, respectively, (12aR)-7-hydroxy-12-[(3,4-difluorophenyl)(pheni)methyl]-3,4,12,12a-tetrahydro-1H-[1,4]oxazino[3,4c]pyrido [2,1-f][1,2,4]triazine-6,8-dione (1.7.2):

LC MS (20 min), m/z 440 (M+1), according to 1H NMR data, a mixture of diastereoisomers 1.8.2 and 1.9.2 in a ratio of 1.5:1;

(12aR)-7 -hydroxy-12-[(10R)-(3,4-difluorophenyl)(pheny)methyl]-3,4,12,12a-tetrahydro-1H[1,4]oxazino[3,4- c]pyrido[2,1-f][1,2,4]triazine-6,8-dione (1.8.2):

LC MS (20 min), Rt=13.99 (220 nm), m/z 440 (M+1),

1H NMR (400 MHz, DMSO) δ 7.89-7.76 (m, 1H), 7.59-7.44 (m, 2H), 7.33-7.13 (m, 6H), 5.74 (s, 1H), 5.44 (d, J=7.6 Hz, 1H), 4.60 (d, J=7.4 Hz, 1H), 4.37 (d, J=12.7 Hz, 1H), 3.96 (d, J=8.3 Hz, 1H), 3.75-3.56 (m, 2H), 3.44-3.37 (m, 2H), 3.22-3.14 (m, 1H); and (12aR)-7-hydroxy-12-[(10S)-(3,4-difluorophenyl)(pheny)methyl]-3,4,12,12a-tetrahydro-1H[1,4]oxazino[3, 4-c]pyrido[2,1-f][1,2,4]triazine-6,8-dione (1.9.2):

LC MS (20 min), Rt=14.32 (220 nm), m/z 440 (M+1),

1H NMR (400 MHz, DMSO) δ 7.66 (d, J=7.4 Hz, 2H), 7.46 (t, J=7.4 Hz, 3H), 7.43-7.32 (m, 2H), 7.28 (dd, J=18.9, 8.6 Hz, 1H), 7.22-7.14 (m, 1H), 5.78 (s, 1H), 5.58 (d, J=7.6 Hz, 1H), 4.52 (dd, J=10.0, 2.8 Hz, 1H), 4.37 ( d, J=12.0 Hz, 1H), 3.92 (dd, J=10.8, 2.8 Hz, 1H), 3.69-3.60 (m, 2H), 3.42-3.35 (m, 1H), 3.03 (t, J=11.1 Hz , 1H).

Example 14 -1H-[1,4]oxazino[3,4-c]pyrido[2,1-f][1,2,4]triazin-7-yl}oxy)methyl methyl carbonate (1.8.4) and ({ (12aR)-12-[(5)-(3,4-difluorophenyl)(pheni)methyl]-7-hydroxy-6,8-dioxo-3,4,6,8,12,12a-hexahydro-1H[ 1,4]oxazino[3,4-c]pyrido[2,1-f][1,2,4]triazin-7-yl}oxy)methyl methyl carbonate (1.9.4).

Obtained by analogy with compounds 1.1.4, 1.2.4 and 1.3.4, the synthesis of which is described in example 7.

Diastereoisomer 1.8.4:

LC MS (20 min), m/z 528 (M+1);

1H NMR (DMSO-d ₆ , 300 MHz, 80°C) δ 7.76 (m, 1H), 7.48 (m, 2H), 7.35 (m, 1H), 7.24 (m, 5H), 5.73 (m, 1H) , 5.66 (m, 3H), 4.57 (m, 1H), 4.37 (m, 1H), 4.00 (m, 1H), 3.75 (s, 3H), 3.71 (m, 1H), 3.43 (m, 1H), 3.30 (m, 1H), 3.10 (m, 1H).

Diastereoisomer 1.9.4:

LC MS (20 min), m/z 528 (M+1);

1H NMR (DMSO-d ₆ , 300 MHz, 80°C) δ 7.65 (m, 2H), 7.43 (m, 5H), 7.21 (m, 2H), 5.79 (d, J=8.0 Hz, 1H), 5.70 (m, 3H), 4.51 (m, 1H), 4.36 (m, 1H), 3.97 (m, 1H), 3.75 (s, 3H), 3.68 (m, 1H), 3.45 (m, 1H), 3.29 ( m, 1H), 2.95 (m, 1H).

Example 15 [3,4-c]pyrido[2,1-e][1,2,4]triazine-6,8-dione (1.10.1) and its diastereoisomers 1.11.1 and 1.12.1.

To a mixture of 400 mg (1.222 mmol, 1 eq.) 7-(benzyloxy)-3,4,12,12a-tetrahydro-1H-[1,4]oxazino[3,4c]pyrido[2,1-f][ 1,2,4]triazine-6,8-dione (2) and 358 mg (1.344 mmol, 1.1 eq.) (3,4-difluorophenyl)[2(methylthio)phenyl]methanol (6) added 1166.5 mg (1.833 mmol, 1.5 eq.) 50% T3P in ethyl acetate and 0.4 ml ethyl acetate. 235 mg (2.444 mmol, 2 eq.) of methanesulfonic acid were added to the suspension and the mixture was heated in a microwave reactor (CEM) MW, 100°C, 5.5 h. 4 ml of water was added to the reaction mixture while cooling with ice and the mixture was stirred in an ice bath for 1 hour. 30 ml of water was added to the reaction mixture and the organic phase was extracted with ethyl acetate (3 times 30 ml). The combined organic layer was washed with saturated NaHCO ₃ solution, brine, dried over Na2SO4 and rotary evaporated to dryness. Product 1.10.1, consisting of its diastereoisomers 1.11.1 and 1.12.1, is partially debenzylated, so the mixture is sent to the next step without purification.

LC MS (3 min): Rt=1.40 (220 nm), m/z 576 (M+1).

Example 16 3,4-c]pyrido[2,1-e][1,2,4]triazine-6,8-dione (1.10.2), consisting of its diastereomers 1.11.2 and 1.12.2.

The product obtained in Example 15 was dissolved in 10 ml of dimethylacetamide and 259 mg (6.11 mmol, 5 eq.) LiCl was added to the solution. The mixture was heated to 80°C and stirred for 3 hours. The reaction mass was evaporated to dryness on a rotary evaporator, 50 ml of 0.5 M HCl was added to the residue, stirred at room temperature for 10 min, and the resulting precipitate was filtered off. The precipitate was washed with water and dried in air. The product is purified by HPLC. Product 1.10.2 (LC MS (20 min): Rt=14.49 (220 nm), m/z486 (M+1)) according to 1H NMR analysis consisted of diastereomers 1.11.2 and 1.12.2 in a ratio of 60:40.

Example 17 [1,2,4]triazine-6,8-dione (1.13.1).

To a mixture of 380 mg (1.2 mmol) 7-(benzyloxy)-3,4,12,12a-tetrahydro-1H-[1,4]oxazino[3,4с]pyrido[2,1-1][1,2, 4]triazine-6,8-dione (2) and 214 mg (1.2 mmol) of diphenylmethanol (7) in 7.4 g (12.0 mmol) 50% T3P in ethyl acetate, 223 mg (2.4 mmol) of methanesulfonic acid were added and stirred at 50°C

- 29 044023

h. The reaction mass was cooled to room temperature, 50 ml of ethyl acetate was added, washed with ml of a saturated NaHCO ₃ solution, dried over Na ₂ SO ₄ and evaporated to dryness on a rotary evaporator.

The resulting product was purified by column chromatography on silica gel with ethyl acetate. After evaporation of the corresponding fractions, 407 mg (71%) of product 1.13.1 were obtained:

LC MS (20 min), Rt=17.10 (220 nm), m/z 494 (M+1);

¹ H NMR (400 MHz, DMSO) δ 7.66 (d, J=7.5 Hz, 2H), 7.55 (d, J=7.3 Hz, 2H), 7.45 (t, J=7.5 Hz, 2H), 7.40-7.28 ( m, 5H), 7.21 (d, J=7.2 Hz, 2H), 7.19-7.09 (m, 3H), 5.66 (s, 1H), 5.57 (d, J=7.7 Hz, 1H), 5.24 (d, J =10.8 Hz, 1H), 5.05 (d, J=10.9 Hz, 1H), 4.53-4.45 (m, 1H), 4.40 (d, J=12.8 Hz, 1H), 3.92 (d, J=8.2 Hz, 1H ), 3.64 (d, J=9.0 Hz, 1H), 3.45-3.37 (m, 1H), 3.23 (t, J=10.9 Hz, 1H), 2.96 (t, J=10.9 Hz, 1H).

Example 18 ,4]triazine-6,8-dione (1.13.2).

To a solution of 360 mg (0.73 mmol) (12aR)-7-(benzyloxy)-12-diphenylmethyl-3,4,12,12a-tetrahydro-1H[1,4]oxazino[3,4-c]pyrido[2, 1-f][1,2,4]triazine-6,8-dione (1.13.1) in 5 ml of dimethylacetamide, 155 mg (3.65 mmol) LiCl was added, the reaction mass was heated to 80°C and stirred for 3 hours. The reaction mass was evaporated to dryness on a rotary evaporator, 50 ml of 0.5 N HCl was added to the residue, and the product was extracted with EtOAc (30 ml each). The combined extract was washed with brine, dried over Na ₂ SO ₄ and evaporated to dryness on a rotary evaporator. Received 62 mg of product 1.13.2:

LC MS (20 min), Rt=12.85 (220 nm), m/z 404 (M+1);

1H NMR (400 MHz, DMSO) δ 7.66 (d, J=7.3 Hz, 2H), 7.45 (t, J=7.3 Hz, 2H), 7.40-7.33 (m, 1H), 7.28 (s, 2H), 7.25 -7.13 (m, 4H), 5.68 (s, 1H), 5.44 (d, J=7.7 Hz, 1H), 4.56 (d, J =8.0 Hz, 1H), 4.38 (d, J=13.0 Hz, 1H) , 3.94 (d, J=9.1 Hz, 1H), 3.72-3.59 (m, 2H), 3.45-3.37 (m, 2H), 3.12-3.00 (m, 1H).

Example 19 ]triazin-7-yl}oxy)methyl methyl carbonate (1.13.4).

Prodrug 1.13.4 was prepared under the conditions described in example 7 by analogy with the preparation of ({(12aR)-12-[(10S)-5,10-dihydrothieno[3,2-c][2]benzothipin-10-yl] - 4,8-dioxo-3,4,6,8,12,12a-hexahydro1H-[1,4]oxazino[3,4-c]pyrido[2,1-f][1,2,4]triazine- 6-yl}oxy)methyl methyl carbonate (1.1.4).

Prodrug 1.13.4:

LC MS (20 min), Rt=12.85 (220 nm), m/z 492 (M+1);

1H NMR (DMSO-d6, 400 MHz) δ 7.67 (d, J=7.6 Hz, 2H), 7.46 (t, J=7.6 Hz, 2H), 7.37 (m, 2H), 7.27 (m, 2H), 7.19 (m, 3H), 5.68 (s, 1H), 5.67 (d, J=6.8 Hz, 1H), 5.63 (d, J=7.6 Hz, 1H), 5.61 (d, J=6.8 Hz, 1H), 4.51 (dd, J1=10.0 Hz, J2=2.8 Hz, 1H), 4.34 (d, J=13.2 Hz, 1H), 3.96 (dd, J1=10.8 Hz, J2=2.4 Hz, 1H), 3.72 (s, 3H ), 3.68 (dd, J1=11.2 Hz, J2=2.8 Hz, 1H), 3.45 (t, J=10.4 Hz, 1H), 3.26 (m, 1H), 3.01 (dt, J1=12.4 Hz, J2=2.8 Hz, 1H).

Example 20 [2,1-f][1,2,4]triazin-6,8-dione (1.14.1).

To a mixture of 0.67 g (3.1 mmol) 7-(benzyloxy)-3,4,12,12a-tetrahydro-1H-[1,4]oxazino[3,4-c]pyrido[2,1f][1,2, 4]triazine-6,8-dione (2) and 1.0 g (3.1 mol) bis(4-fluorophenyl)methanol (8) in 19.4 g (31.0 mmol) 50% T ₃ P in ethyl acetate added 0.59 g (6.2 mmol) methanesulfonic acids and stirred at 80°C for 16 hours. The reaction mass was cooled to room temperature, 150 ml of ethyl acetate was added and washed with 200 ml of a saturated aqueous solution of NaHCO3, dried over Na2SO4 and evaporated to dryness on a rotary evaporator. The resulting product was purified by column chromatography on silica gel with ethyl acetate. After evaporation of the corresponding fractions, product 1.14.1 was obtained:

LC MS (20 min), Rt=15.66 (220 nm), m/z 530 (M=1);

1H NMR (400 MHz, DMSO) δ 7.76-7.67 (m, 2H), 7.55 (d, J=7.1 Hz, 2H), 7.42-7.21 (m, 8H), 6.97 (t, J=8.7 Hz, 2H) , 5.76 (s, 1H), 5.65 (d, J=7.7 Hz, 1H), 5.24 (d, J=10.9 Hz, 1H), 5.06 (d, J=10.8 Hz, 1H), 4.47 (d, J= 7.2 Hz, 1H), 4.40 (d, J=12.6 Hz, 1H), 3.91 (d, J=8.3 Hz, 1H), 3.64 (d, J=9.0 Hz, 1H), 3.42 (t, J=10.5 Hz , 1H), 3.23 (t, J=10.8 Hz, 1H), 3.01 (t, J =11.4 Hz, 1H).

Example 21 2,1-f][1,2,4]triazine-6,8-dione (1.14.2).

Product 1.14.2:

LC MS (20 min), Rt=12.72 (220 nm), m/z 440;

1H NMR (400 MHz, DMSO) δ 11.61 (br.s, 1H), 7.76-7.64 (m, 2H), 7.39-7.18 (m, 5H), 7.03 (t, J=8.7 Hz, 2H), 5.77 ( d, J =11.9 Hz, 1H), 5.52 (d, J=7.6 Hz, 1H), 4.63-4.46 (m, 1H), 4.37 (d, J=13.4 Hz, 1H), 3.92 (d, J=8.1 Hz, 1H), 3.72-3.57 (m, 2H), 3.39 (t, J=10.8 Hz, 1H), 3.11 (t, J=11.1 Hz, 1H);

was obtained under the conditions described in example 17 for compound 1.13.2.

Example 22 f][1,2,4]triazin-7-yl}oxy)methyl methyl carbonate (1.14.4).

Prodrug 1.14.4 was prepared under the conditions described in example 7, by analogy with the preparation of ({(12aR)-12-[(10S)-5,10-dihydrothieno[3,2-c][2]benzothipin-10-yl ]-4,8-dioxo-3,4,6,8,12,12a-hexahydro1H-[1,4]oxazino[3,4-c]pyrido[2,1-f][1,2,4] triazin-6-yl}oxy)methyl methyl carbonate (1.1.4).

Prodrug 1.14.4:

LC MS (20 min), Rt=12.85 (220 nm), m/z 528 (M+1);

- 30 044023 ¹ H NMR (DMSO-d ₆ , 400 MHz) δ 7.73 (m, 2H), 7.39 (d, J=7.6 Hz, 1H), 7.27-7.32 (m, 4H), 7.02 (m, 2H) ,

5.78 (s, 1H), 5.71 (d, J=7.6 Hz, 1H), 5.68 (d, J=6.8 Hz, 1H), 5.61 (d, J=6.8 Hz, 1H), 4.50 (dd, Ji=10.0 Hz, J ₂ =3.2 Hz,

1H), 4.33 (d, J=13.2 Hz, 1H), 3.95 (dd, J1=10.8 Hz, J2=2.8 Hz, 1H), 3.72 (s, 3H), 3.68 (dd, J1=11.2 Hz, J2= 3.2 Hz,

1H), 3.46 (t, J=10.4 Hz, 1H), 3.26 (dt, J1=11.6 Hz, J2=2.0 Hz, 1H), 3.05 (dt, J1=12.4 Hz, J2=3.2 Hz, 1H).

Example 23. In vitro activity of new inhibitors against influenza virus isolates in MDCK cell culture using enzyme-linked immunosorbent assay (ELISA).

Protocol 1 from ImQuest BioSciences (Frederick, Maryland, USA) was used to determine the compound activity of influenza A/California/2009 (H1N1) isolates.

Cell preparation. MDCK cells (canine kidney cells, CCL-34) were obtained from ATCC and passaged in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 1 mM sodium pyruvate and 0.1 MM NEAA, T-75 flasks before use in antiviral assay. On the day prior to analysis, cells were split 1:2 to ensure that they were in exponential growth phase at the time of infection. Determination of cell number and viability was performed using a hemocytometer and trypan blue dye exclusion. Cell viability was greater than 95% for the cells to be used in the assay. Cells were resuspended at 1x104 cells/well in tissue culture medium and added to flat-bottom microtiter plates in a volume of 100 μl. The plates were incubated at 37°C/5% CO2 overnight to ensure cell adhesion.

Preparation of the virus. Influenza virus A/CA/04/09 (NR-13685-) was obtained from BEI Resources (Manassas, VA) and grown in MDCK cells to produce stock virus pools. The pre-titrated aliquot of virus was removed from the freezer (−80°C) and allowed to slowly thaw to room temperature in a biosafety cabinet. The virus was resuspended and diluted in assay medium (DMEM supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 1 mM sodium pyruvate, 0.1 mM NEAA, and 1 μg/ml TPKK-treated trypsin) such that the amount of virus added to each well in a volume of 100 μl was determined to kill 85 to 95% of the cells 4 days postinfection (MOI 0.01).

Tablet format. Each plate contained cell control wells (cells only), virus control wells (cells plus virus), colorimetric drug control wells (drug only), and experimental wells (drug plus cells plus virus). Samples were tested three times for potency with five half dilutions per compound.

Efficacy and toxicity of XTT. After incubation at 37°C in a 5% CO2 incubator, test plates were stained with the tetrazolium dye XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5[(phenylamino)carbonyl]-2H-tetrazolium hydroxide , Sigma-Aldrich). XTT-tetrazolium is metabolized by mitochondrial enzymes of metabolically active cells to a soluble formazan product, allowing rapid quantitative analysis of inhibition of virus-induced cell killing by antiviral test substances. XTT solution was prepared daily as a 1 mg/mL stock in RPMI1640. A solution of phenazine metasulfate (PMS, Sigma-Aldrich) was prepared at a concentration of 0.15 mg/ml in PBS and stored in the dark at -20°C. XTT/PMS stock material was prepared immediately before use by adding 40 μL of PMS per mL of XTT solution. Fifty microliters of XTT/PMS was added to each well of the plate and the plate was reincubated for 4 h at 37°C. The plates were sealed with adhesive plate sealers and gently shaken or inverted several times to mix the soluble formazan product, and the plate was read spectrophotometrically at 450/650 nm using a Molecular Devices Vmax plate reader.

Data analysis. Raw data was collected from Softmax Pro and imported into a Microsoft Excel XLfit4 spreadsheet for analysis using four-parameter fit calculations.

Protocol 2 of the Research Institute of Vaccines and Serums named after. I.I. Mechnikov (FGBNU NIIVS named after I.I. Mechnikov, Moscow) was used to determine the activity of compounds of influenza isolates A/Aichi/2/69 (H3N2) and A/Perth/265/2009 (H1N1pdm09) (H275Y).

To determine the anti-influenza activity of new inhibitors (J and the prototype), MDCK cells were seeded in 96-well Costar plates with an average density of 30,000-35,000 cells per well and grown in Eagle's minimal medium in the presence of 5% fetal calf serum and 10 mM glutamine until a complete monolayer. Before infection with the virus, the cells were washed 2 times with serum-free medium. The test compounds were added to the cells in a 2-fold concentration in 100 μl of minimal Eagle medium. 100 μl of the same medium was added to the viral control, and 200 μl to the cell control. Since they were used human influenza virus strains, study samples and virus dilutions were prepared in a medium supplemented with 2.5 μg/ml TRCK trypsin.After incubation of the cells with the study drugs for 2 hours at 37°C, 100 μl of allantoic acid was added to the wells, excluding the cell control virus diluted in a medium supplemented with 5 μg/ml TRCK trypsin (from 0.1 to 5 PFU per cell).The plates were then incubated for 24 hours in an atmosphere of 5% CO2 at 37°C.

-

Claims (16)

After incubation, the medium was removed and the cells were fixed with 80% acetone in phosphate-buffered saline for 20 min, dried well, and then washed 3 times with phosphate-buffered saline with 0.05% Tween-20 (ELISA solution). These and all further washing procedures were carried out with the specified solution. Next, 100 μl of a phosphate-buffered saline solution with 1% fetal serum and 0.05% Tween-20 was added to the cells and incubated at 37°C for 30 min. After removing the solution, 100 μl of monoclonal antibodies (MAbs) to the internal proteins of the influenza A virus (NP+M1), diluted 1:1000 in an ELISA solution, were added to the cells. After incubation with antibodies for 1 hour at 37°C and subsequent washing 3 times, 100 μl of rabbit anti-mouse IgG labeled with horseradish peroxidase at a dilution of 1:5000 was added to the wells. and incubated for another 1 hour at 37°C. After washing 4 times, bound peroxidase was detected by adding 100 μl of a 0.05% solution of orthophenylenediamine in 0.003% citrate buffer pH 5.0 containing 0.003% H ₂ O _{2 to} the wells. The plates were kept for 15-30 minutes in the dark until color appeared, the reaction was stopped by adding 50 μl of 4N H ₂ SO ₄ , then the optical density (OD) was measured on an automatic spectrophotometer at a wavelength of 450 nm. Wells not infected with virus were used as a cell control. The percentage of inhibition of viral reproduction by the studied compound was determined by the formula Inhibition percentage=100-(OD of experiment - OD of cell control/OD of viral control in the absence of compound - OD of cell control) Four wells of the plate were used for one experimental point, and each value represents the arithmetic mean calculated from this experiment. The concentration of the drug that reduces the OD value by 50% was taken as the inhibitory concentration 50 (IC ₅₀ ). The antiviral activity of the new inhibitors and Baloxavir in influenza A virus isolates in MDCK cell culture is presented in Table. 3. Example 24. Pharmaceutical composition in tablet form. Starch (1700 mg), ground lactose (1700 mg), talc (400 mg) and 1200 mg of prodrug 1.1.4 or 1.11.4 or 1.14.4 were mixed and pressed into a bar. The resulting bar was ground into granules and sifted through a sieve to collect 14-16 mesh granules. The granules thus obtained were formed into tablets of suitable shape, each weighing 80 or 160 mg. Example 25. Pharmaceutical composition in the form of capsules. The prodrug of formula 1.1.4, or 1.11.4, or 1.14.4 was thoroughly mixed with lactose powder in a 2:1 ratio. The resulting powder mixture was packaged in gelatin capsules of suitable size, 36 or 72 mg in each capsule. Industrial applicability The invention can be used in medicine and veterinary medicine. CLAIM 1. Substituted 3,4,12,12a-tetrahydro-1H-[1,4]oxazino[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8-dione general formula 1 ⁰ °\ where R ¹ - 6,7-difluoro-5,10-dihydrothieno[3,2-c][2]benzothipin-10-yl, (3,4-difluorophenyl)(phenyl)methyl, diphenylmethyl, bis( 4-fluorophenyl)methyl; R ² - hydrogen, benzyl or {[(C ₁ -C ₃ -alkyl)oxycarbonyl]oxy}methyl, and its stereoisomer. 2. The compound according to claim 1, which is (12aR)-12-(6,7-difluoro-5,10-dihydrothieno[3,2-c][2]benzothipin-10-yl)-3,4,12 ,12a-tetrahydro-1H[1,4]oxazino[3,4-c]pyrido[2,1-1][1,2,4]triazine-6,8-dione of general formula 1.1; (12aR)-12-[(10S)-6,7-difluoro-5,10-dihydrothieno[3,2-c][2]benzothipin-10-yl]-3,4,12,12a-tetrahydro1H-[ 1,4]oxazino[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8-dione of general formula 1.2; (12aR)-12-[(10R)-6,7-difluoro-5,10-dihydrothieno[3,2-c][2]benzothipin-10-yl]-3,4,12,12a-tetrahydro1H-[ 1,4]oxazino[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8-dione of general formula 1.3: - 32 044023 where R ² has the above meaning. 3. A compound according to claim 1 or 2, which is (12aR)-7-benzyloxy-12-(6,7-difluoro-5,10-dihydrothieno[3,2-c][2]benzothiepin-10-yl) -3,4,12,12atetrahydro-1H-[1,4]oxazino[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8-dione (1.1.1 ); (12aR)-7-benzyloxy-12-[( 10S)-6,7-difluoro-5,10-dihydrothieno[3,2-c][2]benzothipin-10-yl]-3,4,12,12atetrahydro -1H-[1,4]oxazino[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8-dione (1.2.1); (12aR)-7-benzyloxy-12-[(10R)-6,7-difluoro-5,10-dihydrothieno[3,2-c][2]benzothipin-10-yl]-3,4,12,12atetrahydro - 1H-[1,4]oxazino[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8-dione (1.3.1); (12aR)-7 -hydroxy-12-(6,7-difluoro-5,10-dihydrothieno[3,2-c][2]benzothipin-10-yl)-3,4,12,12atetrahydro-III-[ 1,4]oxazino[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8-dione (1.1.2); (12aR)-7-hydroxy-12-[(10S)-6,7-difluoro-5,10-dihydrothieno[3,2-c][2]benzothipin-10-yl]-3,4,12,12atetrahydro -1H-[1,4]oxazino[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8-dione (1.2.2); (12aR)-7-hydroxy-12-[(10R)-6,7-difluoro-5,10-dihydrothieno[3,2-c][2]benzothipin-10-yl]-3,4,12,12atetrahydro -1H-[1,4]oxazino[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8-dione (1.3.2); {[(12aR)-12-(6,7-difluoro-5,10-dihydrothieno[3,2-c][2]benzothipin-10-yl)-6,8-dioxo-3,4,12,12atetrahydro - 1H-[1,4]oxazino[3,4-c]pyrido[2,1-f][1,2,4]triazin-7-yl]oxy}methyl methyl carbonate (1.1.4); ({(12R)-12-[(10S)-6,7-difluoro-5,10-dihydrothieno[3,2-c][2]benzothipin-10-yl]-6,8-dioxo-3,4 ,12,12atetrahydro-1H-[1,4]oxazino[3,4-c]pyrido[2,1-f][1,2,4]triazin-7-yl}oxy)methyl methyl carbonate (1.2.4 ); ({(12aR)-12-[(10R)-6,7-difluoro-5,10-dihydrothieno[3,2-c][2]benzothipin-10-yl]-6,8-dioxo-3,4 ,12,12atetrahydro-1H-[1,4]oxazino[3,4-c]pyrido[2,1-f][1,2,4]triazin-7-yl}oxy)methyl methyl carbonate (1.3.4 ): - 33 044023 4. A compound according to any one of claims 2 or 3, representing (12aR)-7-benzyloxy-12-[(10S)-6,7-difluoro5,10-dihydrothieno[3,2-c][2]benzothepine-10 -yl]-3,4,12,12a-tetrahydro-1H-[1,4]oxazino[3,4-c]pyrido[2,1A[1,2,4]triazine-6,8-dione (1.2 .1) in the form of a solvate with ethanol or isopropanol in the form of orthorhombic crystals, characterized by the following crystallographic data: for the solvate with ethanol, respectively, in a ratio of 4:3 with space group P21212=Z2, a=15.2405(4) A, b=33.4723(8) A, c=11.2776(3) A, α=90.00°, β=90.00° , γ=90.00°, with cell volume V=5753.1(3) A ³ ; for solvate with isopropanol in a corresponding ratio of 8:3 with space group P21212=Z12, a=15.2647(3) A, b=33.3600(7) A, c=11.4164(3) A, α=90.00°, β=90.00° , γ=90.00°, with cell volume V=5813.6(2) A ³ . 5. A compound according to claim 1, which is (12aR)-12-[(3,4-difluorophenyl)(phenyl)methyl]-3,4,12,12a-tetrahydro-1H-[1,4]oxazino[3 ,4-c]pyrido[2,1G][1,2,4]triazine-6,8-dione of general formula 1.7; (12aR)-12-[(R)-(3,4-difluorophenyl)(phenyl)methyl]-3,4,12,12a-tetrahydro-1H-[1,4]oxazino[3,4c]pyrido[2 ,1-f][1,2,4]triazine-6,8-dione of general formula 1.8; (12aR)-12-[(S)-(3,4-difluorophenyl)(phenyl)methyl]-3,4,12,12a-tetrahydro-1H-[1,4]oxazino[3,4с]pyrido[2 ,1-f][1,2,4]triazine-6,8-dione of general formula 1.9: F F F 1.7 1.8 1.9 where R ² has the above meaning. 6. The compound according to claims 1 and 5, which is (12aR)-7-benzyloxy-12-[(3,4-difluorophenyl)(phenyl)methyl]-3,4,12,12a-tetrahydro-1H-[1 ,4]oxazino[3,4c]pyrido[2,1 -f] [1,2,4]triazine-6,8-dione (1.7.1); (12aR)-7-benzyloxy-12-[(R)-(3,4-difluorophenyl)(phenyl)methyl]-3,4,12,12a-tetrahydro-1H[1,4]oxazino[3,4- c]pyrido[2,1-f][1,2,4]triazine-6,8-dione (1.8.1); (12aR)-7-benzyloxy-12-[(S)-(3,4-difluorophenyl)(phenyl)methyl]-3,4,12,12a-tetrahydro-1H[1,4]oxazino[3,4- c]pyrido[2,1-f][1,2,4]triazine-6,8-dione (1.9.1); (12aR)-7 -hydroxy-12-[(3,4-difluorophenyl)(phenyl)methyl]-3,4,12,12a-tetrahydro-1H-[1,4]oxazino[3,4c]pyrido[2 ,1-f][1,2,4]triazine-6,8-dione (1.7.2); (12aR)-7-hydroxy-12-[(R)-(3,4-difluorophenyl)(phenyl)methyl]-3,4,12,12a-tetrahydro-1H-[1,4]oxazino[3,4c ]pyrido[2,1 -f] [1,2,4]triazine-6,8-dione (1.8.2); (12aR)-7-hydroxy-12-[(S)-(3,4-difluorophenyl)(phenyl)methyl]-3,4,12,12a-tetrahydro-1H-[1,4]oxazino[3,4с ]pyrido[2,1-f][1,2,4]triazine-6,8-dione (1.9.2); ({(12aR)-12-[(3,4-difluorophenyl)(phenyl)methyl]-6,8-dioxo-3,4,12,12a-tetrahydro-1H-[1,4]oxazino[3,4с ]pyrido[2,1-1][1,2,4]triazin-7-yl}oxy)methyl methyl carbonate (1.7.4); ({(12aR)-12-[(R)-(3,4-difluorophenyl)(phenyl)methyl]-6,8-dioxo-3,4,12,12a-tetrahydro-1H[1,4]oxazino[ 3,4-c]pyrido[2,1-f][1,2,4]triazin-7-yl}oxy)methyl methyl carbonate (1.8.4); ({(12aR)-12-[(S)-(3,4-difluorophenyl)(phenyl)methyl]-3,4,12,12a-tetrahydro-1H-[1,4]oxazino[3,4c]pyrido [2,1-f][1,2,4]triazin-7-yl}oxy)methyl methyl carbonate (1.9.4): F F F 1.7.1 1.8.1 1.9.1 - 34 044023 1.7.4 1.8.4 1.E.4 7. A compound according to claim 5 or 6, which is (12aR)-7-benzyloxy-12-[(S)-(3,4difluorophenyl)(phenyl)methyl]-3,4,12,12a-tetrahydro-1H- [1,4]oxazino[3,4-c]pyrido[2,1-f][1,2,4]triazine6,8-dione (1.9.1), which is polycrystalline and has a triclinic crystal shape with space group P1=Z1 and lattice parameters a=7.2293(7) A, b=9.5120(9) A, c=10.0046(9) A, α=70.501(2)°, β=85.503(2)°, γ=87.171( 2)°, with cell volume V=646.32(11) A ³ . 8. The compound according to claim 1, which is (12aR)-12-diphenylmethyl-3,4,12,12a-tetrahydro-1H[1,4]oxazino[3,4-c]pyrido[2,1-f] [1,2,4]triazine-6,8-dione of general formula 1.13 About CL about 1.13 where R ² has the above meaning. 9. A compound according to claim 1 or 8, which is (12aR)-7-benzyloxy-12-diphenylmethyl-3,4,12,12a-tetrahydro-1H-[1,4]oxazino[3,4-c]pyrido [2,1f][1,2,4]triazine-6,8-dione (1.13.1); (12aR)-7-hydroxy-12-diphenylmethyl-3,4,12,12a-tetrahydro-1H-[1,4]oxazino [3,4-c]pyrido [2,1f] [1,2,4] triazine-6,8-dione (1.13.2); ({(12aR)- 12-diphenylmethyl-3,4,12,12a-tetrahydro-1H-[1,4]oxazino[3,4-c]pyrido[2,1-f][1,2,4] triazin7-yl}oxy)methyl methyl carbonate (1.13.4): - 35 044023 10. The compound according to claim 1, which is (12aR)-12-[bis(4-fluorophenyl)methyl]-3,4,12,12a-tetrahydro-1H-[1,4]oxazino[3,4-c ]pyrido[2,1 f][1,2,4]triazine-6,8-dione of general formula 1.14 where R ² has the above meaning. 11. A compound according to claim 1 or 10, which is (12aR)-7-benzyloxy-12-[bis(4-fluorophenyl)methyl]-3,4,12,12a-tetrahydro-1H-[1,4]oxazino [3,4c]pyrido[2,1-f][1,2,4]triazine-6,8-dione (1.14.1); (12aR)-7-hydroxy-12-[bis(4-fluorophenyl)methyl]-3,4,12,12a-tetrahydro-1H-[1,4]oxazino[3,4c]pyrido[2,1-f ][1,2,4]triazine-6,8-dione (1.14.2); ({(12aR)-12-[bis(4-fluorophenyl)methyl]-3,4,12,12a-tetrahydro-1H-[1,4]oxazino[3,4-c]pyrido[2,1f][ 1,2,4]triazin-7-yl}oxy)methyl methyl carbonate (1.14.4): 12. Method for preparing compounds of general formula 1 according to any of claims 1-11 by reacting (12aR)7-(benzyloxy)-3,4,12,12a-tetrahydro-1H-[1,4]oxazino[3,4-c ]pyrido[2,1-f][1,2,4]triazine-6,8-dione (2) with 6,7difluoro-5,10-dihydrothieno[3,2-c][2]benzothiepine-10- ol (3), or (3,4-difluorophenyl)(phenyl)methanol (5), or diphenylmethanol (7), or bis(4-fluorophenyl)methanol (8) to obtain the corresponding compounds of formulas 1.1.1-1.3.1 or 1.7.1-1.9.1, 1, 1.13.1, 1.14.1: - 36 044023 with further debenzylation in dimethyl sulfoxide or dimethyl acetamide in the presence of LiCl to obtain the corresponding compound 1.1.2, 1.2.2 and 1.3.2 or 1.7.2-1.9.2 or 1.13.2, 1.14.2 and, if necessary, subsequent reaction with chloromethyl methyl carbonate in dimethylacetamide in the presence of potassium iodide and potassium carbonate to obtain the corresponding compounds of formulas 1.1.4, 1.2.4 and 1.3.4 or 1.7.4-1.9.4, 1.13.4, 1.14.4. 13. Antiviral pharmaceutical composition for the treatment and/or prevention of influenza, containing a compound of general formula 1 where R ¹ is 6,7-difluoro-5,10-dihydrothieno[3,2-c][2]benzothipin-10-yl, (3,4-difluorophenyl)(phenyl)methyl, diphenylmethyl, bis(4-fluorophenyl) )methyl; R ² - hydrogen, benzyl or {[(C ₁ -C ₃ -alkyl)oxycarbonyl]oxy}methyl, or a stereoisomer thereof according to claim 1 in a therapeutically effective amount and a pharmaceutically acceptable excipient. 14. Antiviral pharmaceutical composition according to claim 13, containing a compound of general formula 1.1, 1.2, 1.3, 1.7, 1.8, 1.9, 1.13 or 1.14 according to any of claims 2, 5, 8, 10. 15. Antiviral pharmaceutical composition according to claim 13 or 14, where the compound of formula 1 is selected from compounds 1.1.2, 1.2.2, 1.3.2, 1.7.2, 1.8.2, 1.9.2, 1.13.2, 1.14.2 , 1.1.4, 1.2.4, 1.3.4, 1.7.4, 1.8.4, 1.9.4, 1.13.4, 1.14.4 according to any of paragraphs 3, 4, 6, 7, 9, 11. 16. A method of treating and/or preventing a viral disease, which is influenza, characterized by administering to a patient in a therapeutically effective amount a compound of formula 1 according to any one of claims 1-11 or a pharmaceutical composition of any of claims 13-15 in a therapeutically effective amount. -