Combinations comprising a tricyclic compound and its use in the manufacture of HBV therapeutic agents

Abstract

Regarding a combination comprising a tricyclic compound and its use in the manufacture of a medicament for treating HBV, said combination is a combination of a compound of formula (I) or a pharmaceutically acceptable salt thereof with a drug of any of the following groups a to c: a, hepatitis B surface antigen inhibitor, b, reverse transcriptase inhibitors, c, Hepatitis B surface antigen inhibitor and reverse transcriptase inhibitor. [Formula 1] TIFF2023526346000088.tif27168

Description

Technical Field

[0001] This application claims the priority of Chinese Patent Application CN202010412760.9 with an application date of May 15, 2020, and Chinese Patent Application CN202011633373.4 with an application date of December 31, 2020. This application incorporates the full text of the above-mentioned Chinese patent applications by reference.

[0002] [Technical Field] The present invention relates to a pharmaceutical combination, specifically to a combination comprising a compound having a tricyclic structure or a pharmaceutically acceptable salt thereof, and optionally one or two of a hepatitis B surface antigen inhibitor and / or a reverse transcriptase inhibitor, and to the use of such a combination in the manufacture of a hepatitis B therapeutic agent. Specifically, it discloses a compound of formula (I) or a pharmaceutically acceptable salt thereof, and a combination comprising optionally one or two of a hepatitis B surface antigen inhibitor and / or a reverse transcriptase inhibitor, and the use of such a combination in the manufacture of a hepatitis B therapeutic agent.

Background Art

[0003] Hepatitis B is an inflammatory reaction caused by the invasion of the hepatitis B virus, which may cause a series of problems such as liver pain, enlargement of the liver and spleen, and liver fibrosis, and in severe cases, may cause liver cirrhosis or even liver cancer. According to statistics, there are about 350 million to 400 million hepatitis B virus carriers in the world, and one-third of them are in China. In China, the number of deaths caused by hepatitis B exceeds 500,000 per year.

[0004] At present, there is no specific drug for hepatitis B in the world. The first-choice drugs for hepatitis B treatment in China are mainly nucleoside drugs, interferon, and traditional Chinese medicine, but there are problems such as high cost and inability to cure, so the development of new anti-hepatitis B drugs is essential. The existing single-drug therapy has problems such as drug resistance and less-than-ideal therapeutic effects. The combination use of hepatitis B therapeutic drugs, especially the combination of hepatitis B drugs with different mechanisms, tends to be a research hot spot and a direction for clinical use.

[0005] Patent WO2018153285A1 discloses a compound of formula (I) which is a hepatitis B core protein inhibitor and its use; WO2018214875A1 and WO2018161960A1 respectively disclose a compound of formula (II), a hepatitis B surface antigen inhibitor of a compound of formula (III) and its use. Currently clinically recommended nucleoside or nucleotide reverse transcriptase inhibitors include entecavir, tenofovir disoproxil fumarate, etc.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Patent Document 2

Patent Document 3

Summary of the Invention

Problems to be Solved by the Invention

[0007] The present invention intends to achieve the purpose of synergistically treating HBV through the combination and use of drugs with different mechanisms.

Means for Solving the Problems

[0008] The present invention discloses a pharmaceutical combination, and the combination is a compound of formula (I) or a pharmaceutically acceptable salt thereof, and a pharmaceutical combination of any one of the following groups a to c: a, a hepatitis B surface antigen inhibitor, b, a reverse transcriptase inhibitor, c, a hepatitis B surface antigen inhibitor and a reverse transcriptase inhibitor.

Chemical Formula

[0009] Preferably, here, R a is each independently H, F, Cl, Br, I, NH2, OH, or CN.

[0010] Here, ring A is 5- to 6-membered heterocycloalkyl.

[0011] Here, ring A is tetrahydrofuranyl, tetrahydropyranyl, or dioxanyl.

[0012] Here, ring A is

Chemical Structure

[0013] Here, R1 is H, Cl, F, Br, I, or CH3 optionally substituted with one, two, or three Rs. a More preferably, here, R1 is H, Cl, or CH3.

[0014] Here, R2 is H or CH3.

[0015] R3 is H or CH3.

[0016] Here, L1 is -CH2- or -CH2CH2-.

[0017] In one embodiment, the compound of formula (I) has a structure represented by formula (I-1):

Chemical formula

[0018] Particularly preferred compounds of formula (I) of the present invention are selected from the following specific compounds:

Chemical formula

[0019] In some aspects of the present invention, in the above combination, the hepatitis B surface antigen inhibitor is selected from one of the compounds of formula (II) or a pharmaceutically acceptable salt thereof.

Chemical formula

[0020] In some embodiments of the present invention, in the above combination, the compound of formula (II) is selected from one of the following compounds.

Chemical formula

[0021] In some embodiments of the present invention, in the above combination, the compound of formula (II) is selected from one of the compounds of the following formula. [Chemistry] [Chemistry] [Chemistry]

[0022] Preferably, the hepatitis B surface antigen inhibitor has a structure represented by the following formula (IIa), TIFF0007706475000012.tif36168 Preferably, the hepatitis B surface antigen inhibitor has a structure represented by the following formula (IIb). TIFF0007706475000013.tif33168

[0023] In another embodiment, the hepatitis B surface antigen inhibitor is selected from one of the compounds of the following formula (III) or a pharmaceutically acceptable salt thereof. TIFF0007706475000014.tif44168Here, R1 is selected from H, OH, CN, NH2, or optionally C substituted with 1, 2, or 3 R's 1-5 alkyl, C 1-5 heteroalkyl, C 2-5 alkynyl, C 3-6 cycloalkyl and 3-6 membered heterocycloalkyl, R2 is selected from H, halogen, or optionally C substituted with 1, 2, or 3 R's 1-3 alkyl and C1-3 selected from heteroalkyl, m is selected from 0, 1, 2, 3, 4 and 5, A is selected from phenyl optionally substituted with one, two or three R groups or 5- to 6-membered heteroaryl, R is selected from H, halogen, OH, CN, NH2, =O, CH3, CH3CH2, CH3O, CF3, CHF2, CH2F, said C 1-5 heteroalkyl, 3- to 6-membered heterocycloalkyl, C 1-3 For "hetero" in heteroalkyl, 5- to 6-membered heteroaryl and 3- to 6-membered heterocycloalkyl, each is independently selected from N, -O-, =O, -S-, -NH-, -(C=O)-, -(S=O)-, -(S=O)2-, In any of the above cases, the number of heteroatoms or heteroatomic groups is independently selected from 1, 2 or 3.

[0024] In some embodiments of the present invention, in the above combination, the compound of formula (III) is selected from one of the following compounds.

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0025] In some embodiments of the present invention, in the above combination, the compound of formula (III) is selected from one of the compounds of the following formula.

Chemical formula

Chemical formula

[0026] Preferably, the hepatitis B surface antigen inhibitor has a structure represented by the following formula (IIIa),

Chemical formula

Chemical formula

[0027] In the combination according to the present invention, the reverse transcriptase inhibitor is selected from lamivudine, adefovir dipivoxil, entecavir, tenofovir disoproxil fumarate or tenofovir alafenamide fumarate.

[0028] Preferably, here, the reverse transcriptase inhibitor is selected from entecavir or tenofovir disoproxil fumarate.

[0029] The combination according to the present invention is to produce a pharmaceutical composition by mixing a compound of formula (I) or a pharmaceutically acceptable salt thereof with a medicament of any one of groups a to c as a pharmaceutical active ingredient.

[0030] Here, mixing as the drug active ingredient to produce a pharmaceutical composition means mixing two or three different drugs together as the pharmaceutical active ingredient to produce a combined pharmaceutical composition.

[0031] The combination according to the present invention may be to produce pharmaceutical compositions respectively with a compound of formula (I) or a pharmaceutically acceptable salt thereof and a drug of any one of groups a to c as the pharmaceutical active ingredient respectively, and then separately package and take them separately at the time of taking.

[0032] Here, taking them separately includes taking one or two kinds first, then taking another one or two kinds, and taking two or three kinds simultaneously.

[0033] Use of the combination according to the present invention in the manufacture of a therapeutic drug for hepatitis B virus infection.

[0034] The pharmaceutical preparation composition according to the present invention, which is the combination according to the present invention and at least one pharmaceutically acceptable carrier and / or excipient.

[0035] The present invention further provides a kit comprising the combination according to the present invention or the pharmaceutical preparation composition according to the present invention.

[0036] The present invention further provides the use of the pharmaceutical composition or kit in the manufacture of a drug for treating hepatitis B.

[0037] The method of separately packaging and separately taking when taking the combination of the present invention can be selected from the following methods.

[0038] [Administration method] The following content does not limit the administration method of the combination of the present invention.

[0039] The components of the combination of the present invention can be formulated into pharmaceutical compositions separately, or some or all of them can be formulated into a pharmaceutical composition together. In some embodiments, the combination of the present invention can be formulated into a pharmaceutical composition suitable for single or multiple administrations.

[0040] The components of the combination of the present invention can be administered separately, or some or all of them can be administered together. The components of the combination of the present invention cannot be administered substantially simultaneously, or some or all of them can be administered substantially simultaneously. The components of the combination of the present invention may have the same or different administration cycles.

[0041] The components of the combination of the present invention can each be independently administered by various suitable routes, including oral or parenteral administration (routes such as intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral administrations, for example, administration by injection or infusion), but are not limited thereto. In some embodiments, the components of the combination of the present invention can each be independently administered orally or by injection, for example, intravenous or intraperitoneal injection.

[0042] The components of the combination of the present invention can each be independently in a suitable dosage form, including, but not limited to, tablets, lozenges, pills, capsules (for example, hard capsules, soft capsules, enteric-coated capsules, microcapsules), elixirs, granules, syrups, injections (intramuscular, intravenous, intraperitoneal), granules, emulsions, suspensions, solutions, dispersions and dosage forms of sustained-release preparations for oral or parenteral administration.

[0043] The components of the combination of the present invention can each independently contain a pharmaceutically acceptable carrier and / or excipient. [Advantages of the Invention]

[0044] [Technical Effects] The compound of formula (I) of the present invention is a hepatitis B core protein inhibitor, which can interfere with the viral cccDNA library and inhibit the replication of HBV virus; the compounds of formula (II) and formula (III) of the present invention are hepatitis B surface antigen inhibitors, which can effectively reduce HBsAg. By combining the compound of formula (I) with the hepatitis B surface antigen inhibitor, or by combining the compound of formula (I) with a nucleoside or nucleotide reverse transcriptase inhibitor, or by combining drugs of these three mechanisms, the replication process of HBV virus can be inhibited in multiple channels, and the objectives such as improvement of therapeutic effect, reduction of toxic side effects, shortening of treatment cycle and dosage, and reduction of drug resistance can be achieved, and the reduction of HBV load, the decrease and even removal of HBsAg can be achieved.

[0045] [Definitions and Explanations] Unless otherwise defined, the following terms and phrases used in this specification have the following meanings. A particular term or phrase, if not otherwise defined, should be understood as having its ordinary definition and not being ambiguous or unclear. When a trade name appears in this specification, it refers to the corresponding product or its active ingredient.

[0046] As used in this specification, "pharmaceutically acceptable" applies to those compounds, materials, compositions and / or dosage forms which are within the scope of sound medical judgment, suitable for contact with human and animal tissues, and without excessive toxicity, irritation, allergic reaction or other problems or complications, and commensurate with a reasonable benefit / risk ratio.

[0047] The term "pharmaceutically acceptable salt" refers to salts of the compounds of the present invention, which are prepared with relatively non-toxic acids or bases with compounds having specific substituents found in the present invention. When relatively acidic functional groups are included in the compounds of the present invention, base addition salts can be obtained by contacting these compounds with a sufficient amount of base in a single solution or in a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine or magnesium salts or similar salts. When relatively basic functional groups are included in the compounds of the present invention, acid addition salts can be obtained by contacting these compounds with a sufficient amount of acid in a single solution or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts and organic acid salts, as well as salts of amino acids (such as arginine, etc.) and salts of organic acids such as glucuronic acid. The above inorganic acids include, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate ion, phosphoric acid, monohydrogen phosphate ion, dihydrogen phosphate ion, sulfuric acid, hydrogen sulfate ion, hydroiodic acid, phosphorous acid, etc. The above organic acids include, for example, acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, methanesulfonic acid and similar acids. Some specific compounds of the present invention contain both basic and acidic functional groups and can therefore be converted into any base addition salt or acid addition salt.

[0048] The pharmaceutically acceptable salts of the present invention can be synthesized by conventional methods from parent compounds containing acidic or basic groups. Usually, the production method of such salts is to react these compounds in the form of free acids or bases with a stoichiometric amount of an appropriate base or acid in water, an organic solvent, or a mixture of both.

[0049] The term "pharmaceutical composition" refers to a mixture of one or more active ingredients or their drug combinations described herein and a pharmaceutically acceptable excipient. The purpose of the pharmaceutical composition is to facilitate the administration of the compounds or their drug combinations described herein to a subject.

[0050] The term "pharmaceutically acceptable carrier" refers to any formulation or carrier medium that can deliver an effective amount of the active substance of the present invention, does not interfere with the biological activity of the active substance, and has no toxicity or side effects on the host or patient, including water, oil, vegetables and minerals, cream-based, lotion-based, ointment-based, etc. These bases include suspending agents, tackifiers, penetration enhancers, etc. These formulations are well known to those skilled in the fields of cosmetics or topical pharmaceuticals.

[0051] The term "excipient" generally refers to the carrier, diluent, and / or medium necessary for the formulation of an effective pharmaceutical composition.

[0052] The terms "comprising" or "comprised of" should be understood in an open, non-exclusive sense, i.e., "including but not limited to".

[0053] The term "treatment" means administering the compounds or formulations described herein to prevent, ameliorate, or eliminate a disease or one or more symptoms associated with the disease, including the following: (1) Preventing the occurrence of a disease or disease state in a mammal, particularly when such a mammal is susceptible to the disease state but has not been diagnosed as having the disease state. (2) inhibiting a disease or disease state, i.e., arresting its progression, (3) alleviating a disease or disease state, i.e., regressing the disease or disease state.

[0054] The terms “effective amount” or “therapeutically effective amount” with respect to a medicament or pharmacologically active agent refer to a non-toxic but sufficient amount of the medicament or agent to achieve the desired effect. In the case of the oral dosage forms of the present invention, the “effective amount” of one active substance in the composition refers to the amount necessary to achieve the desired effect when used in combination with another active substance in the composition. The determination of the effective amount varies from person to person and depends on the age and general condition of the subject, and also on the particular active substance, and the appropriate effective amount in an individual case can be determined by one of ordinary skill in the art based on routine experimentation.

[0055] The term “administering” refers to physically introducing a composition containing a therapeutic agent to a subject using any one of a variety of methods and delivery systems known to those of ordinary skill in the art. Routes of administration include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes, for example, administration by injection or infusion. As used herein, the phrase “parenteral administration” generally means a pattern of administration other than enteral and topical administration by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subarticular, subcapsular, subdural, intraspinal, epidural and intracostal injection and infusion, and in vivo electroporation. In some embodiments, the combination is administered by a route other than parenteral, and in some embodiments, it is administered orally. Other non-parenteral routes include topical, epidermal or mucosal administration routes, for example, intranasal, vaginal, rectal, sublingual or topical administration. Administration can also be carried out, for example, once, multiple times, and / or over one or more extended periods.

[0056] The term “subject” is a mammal. In some embodiments, the subject is a mouse. In some embodiments, the subject is a human.

[0057] As used herein, "administered in combination" or "used in combination" means that two or more active substances can each be administered to a subject simultaneously as a single formulation, or each can be administered sequentially in any order as a single formulation.

[0058] The terms "active ingredient", "therapeutic agent", "active substance" or "active agent" refer to a chemical substance effective in treating a target disorder, disease or condition.

[0059] The compounds of the present invention may exist in the form of specific geometric or stereoisomers. The present invention contemplates all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemic mixtures thereof and other mixtures, such as mixtures rich in enantiomers or diastereomers, and all these mixtures are included within the scope of the present invention. Substituents such as alkyl may have other asymmetric carbon atoms. All these isomers and mixtures thereof are included within the scope of the present invention.

[0060] Unless otherwise stated, the terms "enantiomer" or "optical isomer" refer to stereoisomers that are mirror images of each other.

[0061] Unless otherwise stated, the terms "cis-trans isomer" or "geometric isomer" are due to the fact that double bonds or single bonds of ring-constituting carbon atoms cannot rotate freely.

[0062] Unless otherwise stated, the term "diastereomer" refers to stereoisomers in which a molecule has two or more chiral centers and the molecules are in a non-mirror image relationship.

[0063] Unless otherwise stated, "(+)" means dextrorotatory, "(-)" means levorotatory, and "(±)" means racemic.

[0064] Unless otherwise stated, a wedge solid line bond

Chem.

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[0065] The term "substituted" means that any one or more hydrogen atoms at a particular atom are replaced by substituents, and may include deuterium and hydrogen isotopes as long as the particular valence state is normal and the compound after substitution is stable. When the substituent is a keto group (i.e., =O), it means that two hydrogen atoms are substituted. Keto group substitution does not occur in aromatic groups. The term "optionally substituted" means that it may or may not be substituted, and unless otherwise defined, the type and number of substituents are arbitrary as long as they can be chemically stably realized.

[0066] If any of the variables (e.g., R) appears one or more times in the composition or structure of the compound, its definition is independent in any case. Therefore, for example, when one group is substituted with 0 to 2 R's, the above group is optionally substituted with 2 or fewer R's, and in any case, R has independent options. Also, combinations of substituents and / or their isotopes are only allowed if such combinations result in a stable compound.

[0067] When the number of linking groups is 0, for example, -(CRR)0- means that the linking group is a single bond.

[0068] When one of the variables is a single bond, the two groups connected by it are directly connected. For example, when L in A-L-Z represents a single bond, this structure actually becomes A-Z.

[0069] When there is no substituent, it means that the substituent does not exist. For example, when X in A-X does not exist, it means that the structure actually becomes A. When it is not specified through which atom the substituent is substituted for the listed substituents, such substituents can be bonded through any of their atoms. For example, a pyridinyl group as a substituent may be bonded to the substituent through any carbon atom of the pyridine ring.

[0070] When the listed linking group does not specify another linking direction, the linking direction is arbitrary. For example,

Chemical Structure

Chem.

Chem.

[0071] Unless otherwise specified, when a group has one or more bondable sites, any one or more sites of the group can be bonded to other groups by chemical bonds. When the bonding mode of the chemical bond is delocalized and there are H atoms at the bondable sites, when the chemical bond is formed, the number of H atoms at the site decreases to a group with a corresponding valence according to the number of the formed chemical bonds. The chemical bond by which the site is bonded to other groups is a solid straight-line bond

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[0072] Unless otherwise stated, the number of atoms in a ring is generally defined as the number of ring members. For example, "5- to 7-membered ring" refers to a "ring" of 5 to 7 atoms arranged around it.

[0073] Unless otherwise defined, the term "C 1~3 alkyl" represents a saturated hydrocarbon group composed of 1 to 3 straight-chain or branched carbon atoms. The C 1-3 alkyl group includes C 1~2 and C 2~3 alkyl groups, etc., which may be monovalent (e.g., methyl group), divalent (e.g., methylene group), and polyvalent (e.g., methine group). Examples of the C 1~3 alkyl group include, but are not limited to, the methyl group (Me), ethyl group (Et), and propyl group (including n-propyl and isopropyl). Unless otherwise defined, "C 2-8 alkenyl" represents a saturated hydrocarbon group composed of 2 to 8 carbon atoms containing at least one carbon-carbon double bond, and the carbon-carbon double bond may be at any position in the group.

[0074] Unless otherwise defined, the term "4- to 6-membered heterocycloalkyl" by itself or in combination with other terms refers to a saturated cyclic group composed of 4 to 6 ring atoms, where 1, 2, 3, and 4 of the ring atoms are independently heteroatoms selected from O, S, and N, and the remainder are carbon atoms. Here, the nitrogen atom is optionally quaternized, and the nitrogen and sulfur heteroatoms are optionally oxidized (i.e., NO and S(O) p , p is 1 or 2). It includes monocyclic and bicyclic ring systems, where the bicyclic ring systems include spiro rings, fused rings, and bridged rings. Further, with respect to "4- to 6-membered heterocycloalkyl", the heteroatom can occupy the position where the heterocycloalkyl is linked to other parts of the molecule. The 4- to 6-membered heterocycloalkyl includes 5- to 6-membered, 4-membered, 5-membered, and 6-membered heterocycloalkyls, etc. Examples of 4- to 6-membered heterocycloalkyl include azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, tetrahydrothienyl (including tetrahydrothien-2-yl and tetrahydrothien-3-yl, etc.), tetrahydrofuranyl (including tetrahydrofuran-2-yl, etc.), tetrahydropyranyl, piperidinyl (including 1-piperidinyl, 2-piperidinyl, and 3-piperidinyl, etc.), piperazinyl (including 1-piperazinyl and 2-piperazinyl, etc.), morpholinyl (including 3-morpholinyl and 4-morpholinyl, etc.), dioxanyl, dithianyl, isoxazolidinyl, isothiazolidinyl, 1,2-oxazinyl, 1,2-thiazinyl, hexahydropyridazinyl, homopiperazinyl, or homopiperidinyl, but are not limited thereto.

[0075] Unless otherwise indicated, the following terms and phrases used in this specification have the following meanings. A particular phrase or term, if not specifically defined, should be understood as having its ordinary definition and not being uncertain or unclear. When a trade name appears in this specification, it refers to the corresponding product or its active ingredient.

[0076] The intermediate compounds of the present invention can be prepared by various synthetic methods well-known to those skilled in the art, including the specific embodiments listed below, embodiments combined with other chemical synthesis methods, and equivalent alternative methods well-known to those skilled in the art. Preferred embodiments include, but are not limited to, the examples of the present invention.

[0077] The chemical reactions of the specific embodiments of the present invention are completed in a suitable solvent, and the said solvent should be suitable for the chemical changes of the present invention and the reagents and materials required therefor. In order to obtain the compounds of the present invention, it may be necessary for those skilled in the art to modify or select the synthesis process or reaction scheme based on the existing embodiments.

[0078] Hereinafter, the present invention will be specifically described by way of examples, but these examples shall not impose any limitation on the present invention.

[0079] All solvents used in the present invention are commercially available products and may be used as they are without further purification.

[0080] All solvents used in the present invention can be obtained from commercially available products. The present invention uses the following abbreviations: EtOH represents ethanol, MeOH represents methanol, TFA represents trifluoroacetic acid, TsOH represents p-toluenesulfonic acid, mp represents melting point, EtSO3H represents ethanesulfonic acid, MeSO3H represents methanesulfonic acid, THF represents tetrahydrofuran, EtOAc represents ethyl acetate, THF represents tetrahydrofuran, EA represents ethyl acetate, DMAP represents 4-dimethylaminopyridine, DCM represents dichloromethane, and DIPEA represents N,N-diisopropylethylamine.

Brief Description of the Drawings

[0081]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Mode for Carrying Out the Invention

[0082] Hereinafter, the present invention will be specifically described by way of examples, which does not mean any restrictive limitation of the present invention. The present invention has been described in detail herein, and its specific embodiments have also been disclosed. It is obvious to those skilled in the art that various changes and modifications can be made in the specific embodiments of the present invention without departing from the spirit and scope of the present invention.

[0083] Example 1 Production of Compound 1

Chemical Formula

[0084] Synthesis Route:

Chemical Formula

[0085] Step 1: Synthesis of Compound 1-A Add anhydrous dichloromethane (5 L) to a dry 10 L three-necked flask, start stirring, and sequentially add compound 1-SMA (500.00 g) and nitromethane to the three-necked flask. Place the reaction system in a dry ice-ethanol bath and cool it to -10 °C. Control the temperature at -10 °C to 0 °C, slowly add aluminum trichloride (1.15 kg) to the reaction flask, control the temperature below -0 °C, and slowly add α,α-dichlorodimethyl methyl ether (495.00 g) to the reaction kettle. Slowly raise the temperature to room temperature and stir for 18 hours. By TLC (PE:EA 3:1) monitoring, the raw material spot disappeared and a new spot with a large polarity was generated. Withdraw the reaction solution and slowly add it dropwise to a 10% potassium hydrogen sulfate solution (3 L), stir for 20 minutes, and add crash ice to prevent overheating. Transfer the mixed solution to a 25 L separatory funnel, let it stand for separation, separate the dichloromethane layer, and extract the aqueous phase with dichloromethane (2 L × 2). Wash the organic phase with a 10% potassium hydrogen sulfate solution (5 L × 2), separate the organic phase, and dry it with anhydrous sodium sulfate (1 kg). Concentrate the organic phase under reduced pressure to obtain a dark green solid compound 1-A.

[0086] 1 H NMR (400 MHz, deuterated chloroform) δ = 9.97 (br s, 1H), 9.87 - 9.82 (m, 1H), 7.58 (dd, J = 1.5, 3.3 Hz, 1H), 7.36 - 7.29 (m, 1H), 4.37 (q, J = 7.1 Hz, 2H), 1.38 (t, J = 7.2 Hz, 3H).

[0087] Step 2: Synthesis of compound 1-B To a solution of compound 1-A (2 kg, 11.96 mol) in THF (20 L) was added p-toluenesulfonyl hydrazide (2.23 kg, 11.96 mol). The mixture was stirred at 20 °C for about 1 hour. After confirming the disappearance of the starting material by TLC, the reaction system was heated to 60 °C, and then sodium cyanoborohydride (902 g, 14.36 mol) was added in one batch. After the addition was complete, the reaction was heated to 70 °C and stirred for 3 hours. The heating was stopped, and after cooling to room temperature, 5 L of water was added to quench the reaction. Most of the THF was removed under reduced pressure, and the residue was extracted with EA (1.5 L × 3). The organic phases were combined, washed with saturated sodium chloride, and dried over anhydrous sodium sulfate. After filtration, the solvent was removed under reduced pressure, and the crude product was separated by column chromatography to obtain a pale yellow solid compound 1-B.

[0088] Step 3: Synthesis of compound 1-C Methanol (32 L) was added to a 50 L jacketed kettle, and stirring was started. Compound 1-SMB (4000.00 g) and diisopropylethylamine (5.25 L) were sequentially added. The internal temperature was lowered to 5 - 10 °C, and benzyl mercaptan (2490.00 g) was slowly added dropwise while maintaining the internal temperature at 5 - 15 °C. After the addition was complete, the cooling system was closed, and the temperature was allowed to rise naturally. Stirring was continued for 2.5 hours. Stirring was stopped, the rotation speed was adjusted to 100 rpm, the reaction solution was discharged, filtered through a tabletop filter, the cake was washed 3 times with water (5 L), then washed once with EtOH (3 L), and suction filtration was carried out until the cake was no longer viscous to obtain a pale yellow solid compound 1-C.

[0089] Step 4: Synthesis of compound 1-D Add dichloromethane (7.5 L) to a 50 L jacketed kettle, start stirring, add Compound 1-C (1500 g), lower the internal temperature to 0 - 10 °C, and add HCl solution (6 M, 4.12 L). Under the condition of 0 - 10 °C, dropwise add sodium hypochlorite solution (commercially available 8% solution, 23.0 kg) through an opening. After the addition is complete, close the cooling system and continue stirring through the opening for about 17 hours. Then, dropwise add sodium bisulfite solution (1000 g, 5 L aqueous solution) thereto, and detect that there is no oxidant remaining in the aqueous phase with potassium iodide starch test paper. Stop stirring, let it stand for separation, collect the dichloromethane layer, extract the aqueous layer with dichloromethane (2.5 L), and combine the dichloromethane layers. Dry the organic phase over anhydrous sodium sulfate, filter, and remove the solvent under reduced pressure to obtain white solid Compound 1-D.

[0090] 1 H NMR (400 MHz, deuterated chloroform) δ = 8.50 - 8.43 (m, 2H), 8.34 (d, J = 8.2 Hz, 1H), 4.04 (s, 3H).

[0091] Step 5: Synthesis of Compound 1-E Add tetrahydrofuran (10 L) to a dry 50 L jacketed kettle, start stirring, add Compound 1-B (2000 g), and lower the internal temperature to 0 - 10 °C. Maintain the temperature at 0 - 15 °C within about 1.5 hours and add potassium tert-butoxide (1 M THF solution, 15.67 L). After completion, raise the temperature to about 20 °C and continue stirring for 1 hour. Lower the temperature to 0 - 10 °C and slowly add a tetrahydrofuran (10 L) solution of Compound 1-D (4380 g). After the addition is complete, slowly raise the temperature to 15 °C and continue stirring for about 16 hours. Add ethyl acetate (10 L) for extraction, wash the organic phase twice with saturated sodium chloride solution (10 L), combine the aqueous phases, extract with EA (5 L), and combine the organic phases. Remove the solvent from the organic phase under reduced pressure to obtain light yellow solid Compound 1-E.

[0092] 11H NMR (400 MHz, DMSO-d6) δ = 8.55 (d, J = 1.4 Hz, 1H), 8.37 (dd, J = 1.5, 8.3 Hz, 1H), 7.91 (d, J = 8.3 Hz, 1H), 7.60 (s, 1H), 7.13 (d, J = 1.8 Hz, 1H), 4.02 (q, J = 7.0 Hz, 2H), 3.93 (s, 3H), 2.10 (s, 3H), 1.08 (t, J = 7.1 Hz, 3H).

[0093] Step 6: Synthesis of Compound 1-SM1 Compound 1-E (1000.0 g) was added to a dry 10 L three-necked flask, stirring was started, glacial acetic acid (5 L) was added, and the internal reaction temperature was controlled at 25 - 30 °C. Iron powder (1 eq, 140.9 g) was slowly added. After stirring for 30 minutes, the second batch of iron powder (0.5 eq, 70.44 g) was slowly added. After continuing to stir for 30 minutes, the third batch of iron powder (0.5 eq, 70.44 g) was slowly added. After stirring for another 30 minutes, the fourth batch of iron powder (0.5 eq, 70.44 g) was added, and stirring was continued until the raw materials disappeared to allow the reaction to proceed, generating a new spot with a large polarity. Stirring was stopped, and the reaction solution was transferred to a 25 L separatory funnel. 10 L of ethyl acetate was added, and it was washed twice with 5 L of saturated aqueous sodium bisulfate solution, separated, and the aqueous phase was back-extracted with 5 L of ethyl acetate. The organic phases were combined, washed with 10% aqueous NaOH solution until pH > 8, and the organic phase was separated and collected. The organic phase was concentrated under reduced pressure to obtain the white solid compound 1-SM1.

[0094] 1 1H NMR (400 MHz, DMSO-d6) δ = 7.79 - 7.71 (m, 2H), 7.50 (d, J = 1.8 Hz, 1H), 7.14 (dd, J = 1.7, 8.5 Hz, 1H), 6.96 (d, J = 2.0 Hz, 1H), 6.42 (s, 2H), 4.11 (q, J = 7.2 Hz, 2H), 3.84 (s, 3H), 2.04 (s, 3H), 1.16 (t, J = 7.1 Hz, 3H)

[0095] Step 7: Synthesis of Compound 1-F Toluene (12 L) was added to a dry 50 L jacketed kettle, stirring was started, 2-bromoethanol (9930 g) was added, and then boron trifluoride ethyl etherate (268 g) was added. The reaction was heated to 30 - 35 °C. Compound 1-SMC (3500 g) was slowly added dropwise, and the addition was completed in about 1.5 hours. At this time, the internal temperature of the reaction was raised to about 55 - 65 °C, the heater temperature was adjusted to 60 °C, and the internal temperature was maintained at 55 - 65 °C for 1 hour. The internal temperature of the reaction system was lowered to about 10 °C, and an aqueous sodium hydroxide solution (3783 g, 17.5 L of water) at about 20 °C was slowly added to the reaction system while maintaining the internal temperature at 10 - 20 °C. After adding the NaOH solution, the temperature controller was stopped, and the reaction was continuously stirred for about 16 hours. Stirring was stopped, the mixture was allowed to stand, separated, the aqueous layer was extracted with 2-methyltetrahydrofuran (10 L), the organic phases were combined, washed with water (10 L), allowed to stand, separated, and the organic phase was collected. The organic phase was concentrated under reduced pressure to obtain a colorless oily Compound 1-F.

[0096] 1 H NMR (400 MHz, deuterated chloroform) δ = 3.87 - 3.71 (m, 4H), 3.66 - 3.59 (m, 3H), 3.42 (dd, J=6.0, 11.7 Hz, 1H), 3.20 - 3.13 (m, 1H), 2.79 (t, J=4.6 Hz, 1H), 2.65 - 2.59 (m, 1H)

[0097] Step 8: Synthesis of Compound 1-G An aqueous solution of sodium hydroxide (3240 g, 15 L of water) was added to a 50 L jacketed kettle, compound 1-F (4430 g) was added, heating was started, and after the reaction temperature was raised to 90 °C, stirring was continued for 1 hour. Cooling was started and the temperature was lowered to about 15 °C. A tetrahydrofuran solution of p-toluenesulfonyl chloride (6180 g, 15 L of tetrahydrofuran) was added, the temperature controller was closed, and the reaction was stirred at about 15 °C for about 16 hours. Stirring was stopped, the mixture was allowed to stand, separated, the aqueous phase was extracted with 2-methyltetrahydrofuran (10 L), and the 2-methyltetrahydrofuran phase (which had white insoluble matter that disappeared after washing with water) was washed with water (5 L), and the organic phases were combined. DMAP (500 g) and triethylamine (2.5 L) were added to the organic phase, stirred for 30 minutes, washed with saturated sodium chloride solution (10 L), allowed to stand and separated, and the aqueous phase was discarded. The organic phase was washed with potassium hydrogen sulfate solution (3800 g, 15 L of water) and saturated sodium chloride solution (5 L × 2), allowed to stand and separated, and the organic phase was collected. The organic phase was concentrated under reduced pressure to remove the solvent, and the crude product compound 1-G was obtained.

[0098] 1 H NMR (400 MHz, deuterated chloroform) δ = 7.77 (d, J = 8.3 Hz, 2H), 7.34 (d, J = 8.2 Hz, 2H), 4.03 - 3.91 (m, 2H), 3.80 - 3.49 (m, 8H), 3.33 (dd, J = 9.9, 11.4 Hz, 1H), 2.43 (s, 3H)

[0099] Step 9: Synthesis of compound 1-H Acetone (30 L) was added to a clean 50 L jacketed kettle, stirring was started, Compound 1-G (4500 g) was added, then sodium iodide (6190 g) was added, heating was started, and after the reaction temperature was raised to 75 °C, stirring was continued for 16 hours. After cooling to room temperature, filtration was carried out, and the filtrate was concentrated under reduced pressure at 50 °C. Ethyl acetate (15 L) and water (10 L) were added to the concentrated crude product, stirred, allowed to stand and separated, and the organic phase was washed with 0.5 M sodium thiosulfate (10 L). After combining the aqueous phase and the sodium thiosulfate solution, extraction was carried out with EtOAc (5 L). The combined organic phases were washed with saturated sodium chloride solution (10 L), allowed to stand, separated, and the organic phase was collected. The organic phase was concentrated under reduced pressure to remove the solvent, and crude product Compound 1-H was obtained.

[0100] 1 H NMR (400 MHz, deuterated chloroform) δ = 3.90 - 3.83 (m, 2H), 3.81 - 3.75 (m, 2H), 3.74 - 3.65 (m, 5H), 3.63 - 3.49 (m, 7H), 3.31 - 3.18 (m, 3H), 3.06 - 3.04 (m, 2H)

[0101] Step 10: Synthesis of Compound 1-I DMSO (20 L) was added to a clean 50 L jacketed kettle, stirring was started, Compound 1-H (4700 g) was added, the temperature was raised to 35 °C, then sodium cyanide (1010 g) was added, and the internal temperature of the reaction was raised to about 60 °C within 20 minutes, and then gradually lowered to 35 °C, and stirring was continued for about 16 hours. An aqueous sodium bicarbonate solution (2000 g of sodium bicarbonate, 10 L of water) was added to the reaction system, stirring was continued for about 5 minutes, EtOAc:MeOH (20 L, 2 L) was added, stirring was continued for 2 minutes, and allowed to stand for about 1 hour. Separated, about 30 L of the lower layer solution was separated, and extraction was carried out twice with EtOAc:MeOH (15 L:1.5 L for the first time, 5 L:0.5 L for the second time). The upper organic phase after extraction and the upper layer of the remaining reaction solution were combined, washed three times with saturated sodium chloride solution (10 L each), allowed to stand, separated, the aqueous phase was discarded, and the organic phase was collected. The organic phase was concentrated under reduced pressure to remove the solvent, and the crude product was separated by column chromatography to obtain colorless oily Compound 1-I.

[0102] 1 1H NMR (400 MHz, deuterated chloroform) δ = 3.84 - 3.65 (m, 6H), 3.61 - 3.53 (m, 2H), 3.35 (t, J = 10.5 Hz, 1H), 2.49 - 2.44 (m, 2H).

[0103] Step 11: Synthesis of Compound 1-J Under the protection of argon gas, Raney nickel (10.00 g, 116.73 mmol) and EtOH (150 mL) were added to a dry hydrogenation flask. Then, 1-I (20 g, 157.31 mmol) and NH₃·H₂O (13.65 g, 97.36 mmol, 15.00 mL, 25% purity) were added to the reaction system. After replacement, the reaction was stirred at 50 psi and 50 °C for 3.5 hours. The reaction solution was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure to obtain a yellow oily compound 1-J.

[0104] 1 1H NMR (400 MHz, deuterated chloroform) δ = 3.82 - 3.57 (m, 6H), 3.34 - 3.18 (m, 1H), 2.86 - 2.72 (m, 2H), 1.60 - 1.38 (m, 2H).

[0105] Step 12: Synthesis of Compound 1-SM2 1-J (800.00 g) was added to a 5 L three-necked flask, and stirring was started. Ethyl acetate (800 mL) was added within about 0.5 hours. Then, 4 M HCl / EtOAc (1.6 L) was slowly added dropwise to the reaction system until the pH < 5, and the internal temperature was maintained at 5 - 15 °C. The cooling system was closed, and the temperature was raised to room temperature, followed by continuous stirring for 1 hour. Stirring was stopped, and the mixture was filtered through a desktop filter to obtain a cake. The cake was concentrated under reduced pressure (40 - 45 °C) to obtain a crude product. Acetonitrile (2 mL / g) was added to the above product, and the mixture was slurried for 1 hour. It was filtered through a desktop filter, and the cake was collected. The organic solution was removed under reduced pressure to obtain a white solid compound 1-SM2.

[0106] 1 1H NMR (400 MHz, CD3OD) δ = 3.88 - 3.72 (m, 5H), 3.67 - 3.59 (m, 1H), 3.36 - 3.31 (m, 1H), 3.14 (t, J = 6.7 Hz, 2H), 1.87 - 1.67 (m, 2H).

[0107] Step 13: Synthesis of Compound 1-1-A and Compound 1-1-B Toluene (20 L) was added to a dry 50 L jacketed kettle, stirring was started, Compound 1-SM1 (2500 g) was added, and the internal temperature was raised to 30 - 35 °C. The kettle was purged with nitrogen gas to maintain an inert gas atmosphere inside, trimethylaluminum (3.0 L, with a gentle rise in the internal temperature of the kettle upon addition of Al(CH3)3) was added dropwise. After completion, the nitrogen gas was closed, the temperature was raised to 80 - 85 °C, and stirring was continued for about 16 hours. Cooling was started, the reaction temperature was lowered to 20 - 30 °C, about 12 L of half of the reaction solution was transferred, EtOAc (10 L) was added, and they were mixed uniformly. While stirring, the mixed solution was added to a 10% KHSO4 solution (10 L), stirred for 2 minutes, allowed to stand, separated, the organic layer was further washed with a 10% KHSO4 solution (10 L), the aqueous phases were combined, and extraction was performed twice with DCM (7.5 L each). The remaining about 12 L of half of the reaction solution was transferred, and the treatment method was the same as above. The organic phases were combined, the organic phase was concentrated under reduced pressure to obtain a crude product, and twice the volume of n-heptane was added and slurried for 1 hour. It was filtered and dried in vacuo (>12 hours, temperature 40 °C, P ≤ -0.1 MPa). A mixture of Compound 1-1-A and Compound 1-1-B was obtained.

[0108] Step 14: Synthesis of Compound 1-2 Tetrahydrofuran (3840 mL) was added to a 10 L three-necked flask, stirring was started, a mixture of Compound 1-1-A and Compound 1-1-B (480.00 g) was added, and a solution of LiOH·H₂O (118.84 g) in H₂O (960 mL) was slowly added dropwise. After the addition was complete, the temperature was raised to 60 °C and stirred for 1 hour. Concentrated HCl was added to the reaction solution to adjust the pH of the reaction system to 2, and stirring was stopped. It was allowed to stand and separated. The aqueous phase was extracted twice with THF (600 mL), and the organic phases were combined. The organic phase was concentrated under reduced pressure (40 - 45 °C), the solid was slurried with pure water (2 mL / g) for 0.5 hour, filtered, and the cake was dried in vacuo (>12 hours, temperature 40 °C, P ≤ -0.1 MPa) to obtain Compound 1-2.

[0109] 1 H NMR (400 MHz, DMSO-d6) δ = 11.19 (s, 1H), 8.09 (d, J = 8.2 Hz, 1H), 8.00 (d, J = 1.6 Hz, 1H), 7.88 (dd, J = 1.5, 8.3 Hz, 1H), 7.39 (dd, J = 1.2, 2.0 Hz, 1H), 7.01 (d, J = 2.0 Hz, 1H), 2.05 (s, 3H).

[0110] Step 15: Synthesis of Compound 1 DMF (2.25 L) was added to a 5 L three-necked flask, and stirring was started. Compound 1-2 (400.00 g) and HATU (744.83 g) were sequentially added and stirred for 30 minutes. Then, Compound 1-SM2 (229.86 g) was added. Within 1 hour, DIPEA (568.68 mL) was slowly added dropwise at room temperature. After the addition was complete, the mixture was stirred at room temperature for 16 hours. The reaction solution was transferred to a separatory funnel, ethyl acetate (2 L) and pure water (1 L) were added, and the mixture was stirred for 2 minutes. It was allowed to stand, and the aqueous phase was separated. Further, pure water (1 L) was added for washing, the mixture was stirred, allowed to stand, and separated. The combined aqueous phases were extracted 3 times with EtOAc (500 mL), and the organic phases were combined. The organic phase was sequentially washed 2 times with a sodium carbonate solution (1.5 L), 2 times with a potassium hydrogen sulfate solution (1 L), and 2 times with pure water (1 L). The organic phase was concentrated under reduced pressure (40 - 45 °C) to obtain a crude product. Ethyl acetate (2 mL / g) was added to the crude product, and it was slurried for 1 hour. It was filtered to collect the cake, and Compound 1 was obtained.

[0111] 1 H NMR (400 MHz, DMSO-d6) δ = 11.13 (br s, 1H), 8.73 (br t, J = 5.5 Hz, 1H), 8.05 (d, J = 8.2 Hz, 1H), 7.83 (d, J = 1.3 Hz, 1H), 7.74 (dd, J = 1.5, 8.4 Hz, 1H), 7.36 (s, 1H), 6.98 (d, J = 2.0 Hz, 1H), 3.71 - 3.48 (m, 5H), 3.45 - 3.31 (m, 1H), 3.45 - 3.30 (m, 1H), 3.27 - 3.21 (m, 1H), 3.14 (dd, J = 9.9, 11.2 Hz, 1H), 2.03 (s, 3H), 1.53 (q, J = 7.0 Hz, 2H).

[0112] Biological test experiment

[0113] Experimental Example 1: Study on the in vitro combination administration of Compound 1 and Tenofovir disoproxil fumarate (TDF) against HBV inhibitory activity

[0114] 1. Experimental method 1.1 On the first day, HepG2.2.15 cells were seeded in a 96-well cell culture plate at a density of 40,000 cells / well, and then the cells were cultured overnight at 5% CO2 and 37°C.

[0115] 1.2 On the second day, Compound 1 and TDF were each distributed in orthogonal proportion at seven different concentrations (selecting a concentration gradient of about 8×, 4×, 2×, 1×, 0.5×, 0.25×, 0.125× EC 50 ), added to the 96-well plate, and three duplicate wells were set for each combination. The final concentration of DMSO was 0.5%. The detection concentrations of each compound are as shown in Table 1.

[0116] 1.3 On the fifth day, the cell supernatant was discarded, new medium containing the compound was added, and the cells were cultured at 5% CO2 and 37°C for 3 days. On the eighth day, the supernatant of the cell plate after compound treatment was used to extract DNA according to the instructions of the QIAamp 96 DNA Blood Kit (12).

[0117] 1.4 HBV DNA was quantified by qPCR method. HBV plasmid DNA was used as a standard, and the standard HBV plasmid DNA concentration was diluted 7-fold in a 10-fold gradient from 10 7 copies / μL. A standard curve was fitted using the HBV DNA copy number and CT value of each standard, and the HBV DNA copy number in each test sample was calculated.

[0118] The HBV DNA copy number test data were processed using MacSynegy software (Pricard et al., 1990), and the effect parameters of the combined administration of Compound 1 and TDF were analyzed. The cytotoxicity of the compound against HepG2.2.15 cells was detected using the CellTiter-Glo kit. The chemiluminescence intensity (RLU) of each cell well was detected using a multimode microplate reader according to the method in the kit instructions.

[0119] [Table 1]

[0120] 2. Results The inhibitory activity of HBV by the combined administration of compound 1 and TDF in vitro was evaluated using HepG 2.2.15 cells. The summary of the results of the combined administration of the medicine on the inhibitory activity of HBV and cytotoxicity is as shown in Table 2, and the effect diagram of the combined administration is as shown in Figure 1.

[0121] The results of the experiment showed that the synergy index and antagonism index in the 95% confidence interval of the combined administration of compound 1 and TDF in vitro were 13.27 and -1.74, respectively, indicating a positive effect. None of the compounds showed cytotoxicity within the test concentration (see Table 3).

[0122]

Table 2

[0123] Note: Explanation of the combined index of drugs: When the absolute value of the index < 25, that is, it is a positive effect; when the absolute value of the index is in the range of 25 - 50, that is, it is a mild but clear synergistic effect or antagonistic effect; when the absolute value of the index is in the range of 50 - 100, that is, it is a moderate synergistic or antagonistic effect, which may have important significance in vivo effects. When the absolute value of the index > 100, that is, it is a high - degree synergistic effect or antagonistic effect, which is highly likely to have important significance in vivo effects.

[0124]

Table 3

[0125] 3. Conclusions The combined administration of compound 1 and TDF in vitro showed a positive effect on the inhibitory activity of HBV, and the compounds did not show cytotoxicity within the test concentration. The test results supported the clinical combination of compound 1 and TDF in the treatment of chronic HBV - infected patients.

[0126] Experimental Example 2: Evaluation of the in vivo anti-hepatitis B virus efficacy of the test compound using the AAV / HBV mouse model

[0127] Experimental materials

[0128] 1. Animals Male C57BL / 6 mice at 5 weeks of age free of specific pathogens, purchased from SHANGHAI SLAC LABORATORY ANIMAL CO., LTD.

[0129] 2. Solvent and compound Solvent: 10% Solutol aqueous solution

[0130] Test compound: An appropriate amount of Compound 1 was added to the above solvent and vortexed. After that, a uniform suspension of particles was obtained, and the prepared concentrations were 1.0, 3.0, and 10.0 mg / mL. It was stored at 4 °C until use. When Compound 1 was manufactured, it was calculated according to a salt coefficient of 1.0 and a purity of 100%. Tenofovir disoproxil fumarate (TDF) was purchased from Shanghai Panhong Chemical Technology Co., Ltd. An appropriate amount of TDF was weighed and dissolved in physiological saline to prepare a stock solution of 1 mg / mL. It was vortexed until TDF was completely dissolved, dispensed into 1 mL specifications, and stored at -20 °C. Before each administration, 1 mL of the stock solution was collected and diluted 10-fold with physiological saline to a working solution of 0.1 mg / mL for use in the administration of the day.

[0131] Recombinant virus rAAV8-1.3HBV rAAV8-1.3HBV (type D, ayw) was purchased from Beijing FivePlus Molecular Medicine Institute Co., Ltd., 5+MMI, and the batch number was x2018032301, and it was 1×10 12 viral genome (v.g.) / mL. Before the experiment, it was diluted 5×10 with sterile PBS 11It was diluted to v.g. / mL. 200 μL was injected into each mouse, that is, 1×10 11 v.g. was injected into each mouse.

[0132] 3. Test method Construction of AAV / HBV mouse model AAV / HBV injection: rAAV8-1.3HBV was prepared into a solution with a concentration of 1×10 11 v.g. / 200 μL using sterile PBS in advance before injection.

[0133] Detection of infection level: On the 14th and 21st days after virus injection, ~120 μL of blood was collected from the submandibular vein of all infected mice for serum collection. After incubating the whole blood in a 37°C incubator for 30 minutes, it was centrifuged at 13,200×g for 3 minutes at 4°C to collect ~30 μL of serum. The serum was stored at -80°C and used for the detection of HBV DNA, HBeAg, and HBsAg.

[0134] Grouping: On the 28th day after virus injection, the groups were divided based on the levels of HBV DNA, HBsAg, and HBeAg in the serum samples on the 14th and 21st days after virus injection, and the mouse body weight.

[0135] Definition of experimental days: The first administration day was defined as experimental day 0.

[0136] 4. In vivo experiment design The administration and sampling scheme for the in vivo experiment is as shown in Table 4.

[0137]

Table 4

[0138] Production of serum samples: After culturing the blood samples at 37°C for ~30 minutes, they were centrifuged at 4°C and 13,200 g for 3 minutes, and the separated supernatant was rapidly frozen with dry ice.

[0139] Manufacture and pretreatment of plasma samples: After anticoagulating the blood sample with K2EDTA, it was centrifuged at 4°C and 7,000 g for 10 minutes to separate the supernatant. A precipitant was added to the separated plasma at a ratio of 1:20, [methanol:acetonitrile (v:v, 50:50) solution], vortexed and mixed, and then rapidly frozen with dry ice.

[0140] Recording of body weight: During the in-vivo experiment, the state of the mice was observed regularly, and the body weight of the mice was recorded on the day when infection, administration, blood collection, and the experiment ended.

[0141] Serum was collected by cardiac puncture for use in HBV DNA detection to detect the HBV RNA levels in Groups 1 and 4.

[0142] 5. Analysis of samples The HBsAg content in mouse serum was detected using the HBsAg ELISA (Antu Bio, CL 0310) kit instructions.

[0143] The HBeAg content in mouse serum was detected using the HBeAg ELISA kit (Antu Bio, CL 0312) instructions.

[0144] The HBV DNA content in mouse serum and liver was detected by quantitative PCR.

[0145]

Table 5

[0146] Data analysis: The data was presented as the mean ± standard error of the samples in each group. Unless otherwise specified, for Groups 1 - 6: n = 8. Statistical analysis was performed using the Student’s t test.

[0147] 6. Results 1) Effect of the test compound on serum HBV DNA in the AAV / HBV mouse experiment The HBV DNA content in the sera of mice in each group is as summarized in Figure 2. The serum HBV DNA content in the mice of the solvent group (Group1) maintained stability after administration. In the single-agent group of Compound 1, compared with the solvent group (Group1), the HBV DNA content in the sera of mice in the three dose groups of low (10 mpk, Group2), medium (30 mpk), and high (100 mpk) of Compound 1 began to show a dose-dependent decrease 3 days after administration. The low and medium dose groups maintained stability of the HBV DNA content 7 days after administration. In the high dose group, the HBV DNA content in the serum decreased continuously from 7 to 28 days after administration, and was significantly lower than that of the solvent group (p < 0.01).

[0148] In the combined administration group of Compound 1 and TDF (Group6, 10 + 1 mpk), compared with the solvent group (Group1), the HBV DNA content in the mouse serum decreased 3 days after administration, and the HBV DNA content in the serum maintained stability from 7 to 28 days after administration. Compared with the single-agent groups of Compound 1 (Group2, 10 mpk) and TDF (Group5, 1 mpk), the combined administration showed an effect of significantly decreasing the HBV DNA content in the mouse serum (p < 0.01).

[0149] 2) Effect of the test compound on HBV DNA in the liver in the AAV / HBV mouse experiment The HBV DNA content in the livers of mice in each group is as summarized in Figure 3. Compared with the solvent group (Group1), 28 days after administration, the HBV DNA content in the livers of mice in the three dose groups of low (10 mpk), medium (30 mpk), and high (100 mpk) of Compound 1 decreased. In the combined administration group of Compound 1 and TDF (Group6, 10 + 1 mpk), 28 days after administration, the HBV DNA content in the liver was significantly lower than that of the 10 mpk single-agent group of Compound 1 (p < 0.001).

[0150] 3) Effect of the test compound on serum HBV RNA in the AAV / HBV mouse experiment The HBV RNA content in the sera of mice in each group is as summarized in Figure 4. Compared with the solvent group (Group1), the HBV RNA content in the mouse serum of the high (100 mpk) dose administration group of Compound 1 decreased 28 days after administration.

[0151] 4) Pharmacokinetic analysis of the test compound in AAV / HBV model mice The drug concentrations in the mouse sera of the second group are as summarized in Figure 5 and Table 6. The average values of the drug concentrations in the mouse sera at 0 hours (before administration), 1, 4, 8, 9, 12, and 24 hours after the first administration on the 27th day were 78, 122200, 1630, 605, 10300, 1280, and 66 nM, respectively. The drug absorption reached a peak at 1 hour after administration, the plasma half-life was 2.23 hours, and the AUC 0-inf was 47600 nM·h.

[0152]

Table 6

[0153] 5) Monitoring of the health and body weight of mice During the experiment, the health status and body weight of the mice were monitored regularly. Comparing with the body weight on the 0th day as a reference, the results of the body weight changes of the mice are as summarized in Figure 6. During the administration period, the body weights of all the mice were stable, there was no significant decrease, and the state of the mice was good.

[0154] Note: The body weight changes of the mice were recorded from -28 days to 28 days (the first administration day was set as the 0th day). Comparing with the body weight on the 0th day as a reference, according to the regulations of IACUC, a 20% body weight loss was set as the humane endpoint, and when the body weight of the mice decreased by more than 20%, it was necessary to remove them from the experiment.

[0155] 7. Conclusion Compared with the solvent control group, the HBV DNA in the serum and liver of mice after treatment with Compound 1 decreased significantly, showing a dose-dependent trend in the serum. After the combination of Compound 1 and TDF, the effect was significantly better than that of Compound 1 alone treatment, showing a good combined administration effect. During the experimental period, the body weight of the mice did not decrease significantly, indicating that the mice had good tolerance to the test compound.

[0156] 8. Description In the above experiment, TDF was replaced with ETV (entecavir), and the in vivo anti-viral effect of the combination of Compound 1 and ETV was obtained. The HBV DNA in the mouse serum and liver decreased significantly, which was superior to the treatment with Compound 1 alone.

[0157] Experimental Example 3: Study on the HBV inhibitory activity by the in vitro combination of Compound 1 and the compounds of the following formula (IIb) and formula (IIIb)

Chemical formula

[0158] 1. Experimental materials Cell line: The HepG2.2.15 cells were constructed and provided by WuXi AppTec. The cell medium was DMEM / F12 medium supplemented with 2% fetal bovine serum, 2 mM glutamine, 1× non-essential amino acids, 100 U / mL penicillin and 100 μg / mL streptomycin.

[0159] Reagents: The main reagents used in this study included FastStart Universal Probe Master (Roche, catalog number 04914058001), DNA extraction kit (Qiagen, catalog number: 51162), and CellTiter-Glo (Promega-G7573).

[0160] Equipment: The main equipment used in this study was the 7900 Real-Time PCR instrument (Applied Biosystems), the multi-functional microplate reader (BioTek, Synergy2), and the QuantStudio TM 6Flex System (Applied Biosystems).

[0161] Test compound: Compound 1, the compound of formula (IIb), the compound of formula (IIIb).

[0162] 2. Experimental methods

[0163] 2.1 Anti-HBV activity of single compounds

[0164] 2.1.1 On the first day, HepG2.2.15 cells were seeded in a 96-well cell culture plate at a density of 60,000 cells / well, and then the cells were cultured overnight at 5% CO2 and 37°C. On the second day, Compound 1, the compound of formula (IIb), and the compound of formula (IIIb) were diluted and added to the 96-well plate. Three duplicate wells were set for each combination, and the cells were cultured for 3 days under the conditions of 5% CO2 and 37°C. The detection concentrations of each compound are as shown in Table 7. On the fifth day, the medium containing the compound was replaced with a new one, and the supernatant was collected on the eighth day. HBsAg in the supernatant was detected by ELISA, and at the same time, DNA in the supernatant was extracted, and the content of HBV DNA in the supernatant was measured using quantitative PCR. After collecting the cell supernatant, CellTiter-Glo was added to detect the cell viability.

[0165]

Table 7

[0166] 2.2 Anti-HBV activity of combined compounds

[0167] 2.2.1 On the first day, HepG2.2.15 cells were seeded in a 96-well cell culture plate at a density of 60,000 cells / well, and then the cells were cultured overnight under the conditions of 5% CO2 and 37°C. On the second day, cells were treated by adding compounds at different concentrations. The compounds were diluted in a gradient, and three duplicate wells were set for parallel measurement with a combination of 7×7 concentrations. The compound concentrations for the combination activity test were based on the EC 50 values of each compound. The seven concentration gradients were approximately 2×, 1×, 1 / 2×, 1 / 4×, 1 / 8×, 1 / 16×, and 1 / 32× EC 50 respectively. The compound concentrations for the combination activity test experiment were as shown in Table 8, and the arrangements for combination administration were as shown in Table 9. On the fifth day, the medium was replaced with fresh medium containing the compounds. On the eighth day, the culture supernatant was collected, DNA was extracted, and the HBV DNA content was measured by quantitative PCR. At the same time, cell viability was detected with CellTiter Glo reagent, and the combination effect of the two drugs was analyzed using MacSynergy software.

[0168]

Table 8

[0169]

Table 9

[0170] Note: a = Compound 1, b = the compound of formula (IIb) or the compound of formula (IIIb), 0 represents the absence of the compound, and 1 - 7 represent seven concentrations.

[0171] 3. Experimental results: Using HepG2.2.15 cells, the inhibitory activities of the combination administration in vitro of Compound 1 and the compound of formula (IIb), and Compound 1 and the compound of formula (IIIb) against HBV were evaluated. The inhibitory activities and cytotoxicities of the single compounds against HBV are as summarized in Table 10, and the results of the inhibitory activities and cytotoxicities of the combination administration against HBV are as summarized in Tables 11 - 13.

[0172]

Table 10

[0173]

Table 11

[0174] Note: Explanation of the drug combination index: When the absolute value of the index < 25, that is, it is a positive effect; when the absolute value of the index is in the range of 25 - 50, that is, it shows a mild but clear synergistic or antagonistic effect; when the absolute value of the index is in the range of 50 - 100, that is, it shows a moderate synergistic or antagonistic effect and may have important significance for the in vivo effect. When the absolute value of the index > 100, that is, it shows a high degree of synergistic or antagonistic effect and is highly likely to have important significance for the in vivo effect.

[0175]

Table 12

[0176]

Table 13

[0177] 4. Experimental conclusions In the HepG2.2.15 in vitro HBV-infected model, the test compound inhibited HBV DNA in a dose-dependent manner. The combination of Compound 1 with the compound of formula (IIb) and the combination of Compound 1 with the compound of formula (IIIb) showed positive combination results and did not show cytotoxicity within the combination test concentrations. The test results supported the clinical combination of Compound 1 with hepatitis B surface antigen inhibitors such as the compound of formula (IIb) and the compound of formula (IIIb) in the treatment of chronic HBV infection.

[0178] 5. Explanation According to the above embodiments, Compound 1 is used as Compound 2

Chemical formula

Claims

1. A compound of formula (I) 【Chemical 1】 or a pharmaceutically acceptable salt thereof, and any one of the following groups a to c:[[]] a. A hepatitis B surface antigen inhibitor b. A reverse transcriptase inhibitor c. A hepatitis B surface antigen inhibitor and a reverse transcriptase inhibitor A pharmaceutical composition comprising the same. (Herein, the compound of formula (I) is one of the following compounds 【Chemical Formula 2】 and the hepatitis B surface antigen inhibitor is one of the following compounds [Chemical Formula 3] and the reverse transcriptase inhibitor is selected from lamivudine, adefovir dipivoxil, entecavir, tenofovir disoproxil fumarate or tenofovir alafenamide fumarate.)

2. The composition according to claim 1, wherein the reverse transcriptase inhibitor is selected from entecavir and tenofovir disoproxil fumarate.

3. A medicament for the treatment of hepatitis B virus infection, comprising the composition according to claim 1.

4. A pharmaceutical composition, characterized by comprising the composition according to claim 1 and at least one pharmaceutically acceptable carrier and / or excipient.

5. A kit, characterized by comprising the composition according to claim 1 or the composition according to claim 4.

6. A medicament for the treatment of hepatitis B, comprising the composition according to claim 4 or the kit according to claim 5.

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