CN116178367A - Application of benzoheterocycle compounds in treatment or prevention of hepatitis B - Google Patents

Abstract

The application provides the application of benzoheterocyclic compounds in treating or preventing hepatitis B. The present application also provides the use of the compound of general formula 1, its stereoisomer or pharmaceutically acceptable salt thereof in the preparation of medicines for treating or preventing hepatitis B. The present invention also provides a pharmaceutical composition for treating or preventing hepatitis B, which comprises a compound of general formula 1, its stereoisomer or a pharmaceutically acceptable salt thereof, optionally one or more additional A therapeutic agent or a preventive agent, and a pharmaceutically acceptable carrier.

Description

Application of benzoheterocyclic compounds in the treatment or prevention of hepatitis B

technical field

The application relates to the field of anti-hepatitis B drugs, in particular to the application of benzoheterocyclic compounds, their stereoisomers or pharmaceutically acceptable salts thereof in drugs for treating or preventing hepatitis B.

Background technique

Hepatitis B is an infectious disease mainly caused by hepatitis B virus (HBV). Clinically, the main manifestations are loss of appetite, nausea, upper abdominal discomfort, pain in the liver area, and fatigue. Some patients may have jaundice, fever and hepatomegaly with liver function damage. Some patients can become chronic and even develop into liver cirrhosis, and a few can develop into liver cancer.

The treatment of hepatitis B is a long-term process, and the goal of treatment is to suppress or eliminate HBV to the greatest extent, reduce inflammation and necrosis of liver cells and liver fibrosis, delay and prevent disease progression, reduce and prevent liver decompensation, cirrhosis, Hepatocellular carcinoma and its complications can lead to improved quality of life and prolonged survival.

Currently, there are many hepatitis B therapeutic drugs on the market, mainly through the use of interferon or nucleoside analogues for antiviral treatment. For interferon, recombinant DNA leukocyte interferon (IFN-α) can inhibit the replication of HBV. However, when interferon is used to treat hepatitis B, it is often accompanied by strong adverse reactions, including bone marrow suppression, affecting thyroid function and depression.

Nucleoside analogs inhibit HBV production mainly by inhibiting the reverse transcriptase activity during HBV replication. Clinically available drugs include: lamivudine, famciclovir, acyclovir, adefovir, entecavir, tenofovir, Foscarnet sodium, etc., these drugs have a certain inhibitory effect on HBV.

Although these reverse transcriptase inhibitors can effectively reduce the level of HBV DNA and enable patients to control the level of hepatitis B virus, because their target is the process of reverse transcription of RNA into DNA, they have no direct effect on the clearance of HBV cccDNA and HBsAg. Therefore, the probability of HBsAg seroconversion in nucleoside analog monotherapy is extremely low, and it cannot really cure hepatitis B. Patients need to take drugs for a long time or even lifelong.

Under the condition of taking the above-mentioned drugs for a long time, problems such as drug resistance, huge medical expenses, and serious side effects of drugs are a heavy burden for hepatitis B patients. The point is that there is still no drug that can completely clear the virus and achieve a functional cure for hepatitis B. Therefore, there is an urgent need in this field to provide a new drug for treating hepatitis B that can eliminate HBsAg and achieve functional cure.

Contents of the invention

The application provides the use of benzoheterocyclic compounds in the prevention or treatment of hepatitis B, the compound can reduce the hepatitis B virus (HBV) load, HBsAg level and/or HBeAg level, and even can eliminate HBsAg and HBeAg , is expected to functionally cure hepatitis B and clear hepatitis B virus.

In one aspect, the application provides a benzoheterocyclic compound or a stereoisomer or a pharmaceutically acceptable salt thereof, the compound has a structure shown in Formula 1 (the compound is also herein known as the compound of general formula 1):

in:

X is CH or N;

Y is O, S or NH;

R ₁ represents a 5-10 membered heteroaryl optionally substituted by one or more substituents selected from halogen, C _1-4 alkyl, halogenated C _1-4 alkyl, the heteroaryl comprising 1 to 3 heteroatoms selected from N, O and S;

Each _R independently represents halogen, C _1-4 alkyl, haloC _1-4 alkyl; and

n is an integer from 0 to 4,

Provided that said compound is not:

In one embodiment, Y in Formula 1 is NH.

In _one embodiment, R in the general formula ₁ represents an _8-10 membered Bicyclic heteroaryl containing 1 to 3 heteroatoms selected from N, O and S.

In one embodiment, R in formula ₁ is selected from:

In one embodiment, the compound is selected from:

In another aspect, the present application provides a pharmaceutical composition, which comprises a compound having a structure represented by general formula 1 or a stereoisomer or a pharmaceutically acceptable salt thereof, and optionally one or more Additional therapeutic or prophylactic agents, and pharmaceutically acceptable carriers:

in:

X is CH or N;

Y is O, S or NH;

R ₁ represents a 5-10 membered heteroaryl optionally substituted by one or more substituents selected from halogen, C _1-4 alkyl, halogenated C _1-4 alkyl, the heteroaryl comprising 1 to 3 heteroatoms selected from N, O and S;

Each _R independently represents halogen, C _1-4 alkyl, haloC _1-4 alkyl; and

n is an integer of 0 to 4.

In one embodiment, the compound contained in the pharmaceutical composition is selected from:

In one embodiment, the additional therapeutic or prophylactic agent included in the pharmaceutical composition is at least one selected from interferon, pegylated interferon, or nucleoside analogs. In one embodiment, the nucleoside analogue is selected from entecavir, tenofovir disoproxil fumarate and tenofovir alafenamide.

In yet another aspect, the application provides a compound having a structure represented by general formula 1 or a stereoisomer thereof or a pharmaceutically acceptable compound thereof in the preparation of a medicament for treating hepatitis B:

in:

X is CH or N;

Y is O, S or NH;

R ₁ represents a 5-10 membered heteroaryl optionally substituted by one or more substituents selected from halogen, C _1-4 alkyl, halogenated C _1-4 alkyl, the heteroaryl comprising 1 to 3 heteroatoms selected from N, O and S;

Each _R independently represents halogen, C _1-4 alkyl, haloC _1-4 alkyl; and

n is an integer of 0 to 4.

In one embodiment of said use, said compound is selected from:

In one embodiment of the use, the medicament can reduce the load of hepatitis B virus (HBV) and/or the level of HBsAg and/or the level of HBeAg, even clear the level of HBsAg and/or HBeAg.

In one embodiment of the use, the medicament is capable of reducing HBsAg levels and HBeAg levels simultaneously.

In one embodiment of the use, the medicament is capable of simultaneously clearing HBsAg levels and HBeAg levels.

In one embodiment of said use, said medicament further comprises one or more additional therapeutic or prophylactic agents. In one embodiment, the one or more additional therapeutic or prophylactic agents are selected from at least one of interferons, pegylated interferons, and nucleoside analogs. In one embodiment, the nucleoside analogue is selected from entecavir, tenofovir disoproxil fumarate and tenofovir alafenamide.

In some embodiments, the pharmaceutically acceptable salts of the compound of general formula 1 or its stereoisomer described in this application include but not limited to: acetate, adipate, alginate, aspartic acid Salt, Benzoate, Besylate, Bisulfate, Butyrate, Citrate, Camphorate, Camphorsulfonate, Cyclopentane Propionate, Digluconate, Lauryl Sulfate Salt, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethane Sulfonate, lactate, malate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, thiocyanate, tosylate and undecane salt.

In some embodiments, the compound of the general formula 1 or its stereoisomer or pharmaceutically acceptable salt thereof, the pharmaceutical composition or the drug comprising it is administered by a route selected from the following: oral, rectal, orally Nasal, pulmonary, topical, buccal and sublingual, vaginal, parenteral, subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural.

In some embodiments, the compound of general formula 1 or its stereoisomer or pharmaceutically acceptable salt thereof, a pharmaceutical composition or a drug comprising it described in this application is administered orally.

In some embodiments, the compound of general formula 1 or its stereoisomer or pharmaceutically acceptable salt thereof, the pharmaceutical composition or medicine comprising it described in this application is an oral preparation. In some embodiments, the compound of general formula 1 or its stereoisomer or pharmaceutically acceptable salt thereof described in the present application, the pharmaceutical composition or medicine comprising it is in the form of tablet or capsule.

The technical solution of the present invention has the following beneficial effects:

1. Applying the compound of general formula 1, its stereoisomer or pharmaceutically acceptable salt thereof to treat or prevent hepatitis B, thus providing a novel treatment option for hepatitis B.

2. The compound of general formula 1, its stereoisomer or its pharmaceutically acceptable salt can effectively reduce the hepatitis B virus (HBV) load, HBsAg level and/or HBeAg level at the same time, and can even eliminate HBsAg and HBeAg, It is expected to achieve the effect of functionally curing hepatitis B.

3. The compound of general formula 1, its stereoisomer or pharmaceutically acceptable salt thereof can be combined with one or more other therapeutic agents or preventive agents for the treatment of hepatitis B, especially with the ability to reduce The combination of drugs with viral titers but unable to completely eliminate the virus and reduce the level of HBsAg and/or HBeAg can clear the hepatitis B virus from different aspects and have the possibility of synergistic effect, thus providing a basis for subsequent combination drug design broad mind.

4. The compound of general formula 1, its stereoisomers or pharmaceutically acceptable salts thereof have low cytotoxicity, high cell viability, low toxic and side effects on human body or animals, and high safety.

Description of drawings

Figure 1 is the results of cell viability determined for different concentrations of compound CP15.

Figure 2 shows the inhibition results of different concentrations of compound CP15 on HBV DNA.

Figure 3 shows the inhibition results of different concentrations of compound CP15 on HBeAg.

Figure 4 shows the inhibition results of different concentrations of compound CP15 on HBsAg.

Detailed ways

The inventors of the present application unexpectedly found that the compound of general formula 1 described in the present application, its stereoisomer or its pharmaceutically acceptable salt has the potential activity of inhibiting hepatitis B virus, especially has the effect of reducing hepatitis B virus (HBV) load, HBsAg level and/or HBeAg level, and even the effect of clearing HBsAg and HBeAg, can be used to prevent and treat hepatitis B, and has low cytotoxicity and high safety.

The compound of general formula 1, its stereoisomer or its pharmaceutically acceptable salt

The application provides a group of benzoheterocyclic compounds, their stereoisomers or pharmaceutically acceptable salts thereof, the benzoheterocyclic compounds have the structure shown in the following general formula 1:

in:

X is CH or N;

Y is O, S or NH;

R ₁ represents a 5-10 membered heteroaryl optionally substituted by one or more substituents selected from halogen, C _1-4 alkyl, halogenated C _1-4 alkyl, the heteroaryl comprising 1 to 3 heteroatoms selected from N, O and S;

Each _R independently represents halogen, C _1-4 alkyl, haloC _1-4 alkyl; and

n is an integer of 0 to 4.

In one embodiment, X in the general formula 1 is CH. In one embodiment, X in the general formula 1 is N.

In one embodiment, Y in the general formula 1 is NH. In one embodiment, Y in the general formula 1 is O. In one embodiment, Y in the general formula 1 is S.

In one embodiment, X in the general formula 1 is CH, and Y is NH, then the benzoheterocyclic compounds can be called indole compounds.

In one embodiment, X in the general formula 1 is N, and Y is NH, then the benzoheterocyclic compound can be called a benzo[d]imidazole compound.

In one embodiment, R in the general formula ₁ represents optionally replaced by one or more, preferably one or two, selected from halogen, C _1-4 alkyl, halogenated C _1-4 alkyl Substituent-substituted 5-10 membered heteroaryl containing 1 to 3 heteroatoms selected from N, O and S.

In one embodiment, R in the general formula ₁ represents optionally replaced by one or more, preferably one or two, selected from halogen, C _1-4 alkyl, halogenated C _1-4 alkyl An 8-10 membered bicyclic heteroaryl group substituted by a substituent, the heteroaryl group comprising 1 to 3 heteroatoms selected from N, O and S.

In one embodiment, R in the general formula ₁ represents optionally replaced by one or more, preferably one or two, selected from halogen, C _1-4 alkyl, halogenated C _1-4 alkyl Substituted thienyl, furyl, pyrrolyl, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, oxadiazolyl, triazolyl, tetrazolyl, pyridine Base, pyrimidinyl, pyridazinyl, pyrazinyl, triazinyl, benzimidazolyl, benzopyrazolyl, benzothiazolyl, benzothienyl, imidazopyridyl, thienopyridyl, thieno Pyrimidinyl, thienopyrazinyl, thienopyridazinyl, thiazolopyridyl, isothiazolopyridyl, furopyrimidinyl, furopyridazinyl, triazolopyridyl, imidazopyrimidinyl, imidazo Pyrazinyl, imidazopyridazinyl, indolyl, indolyl, indazolyl, isoindazolyl, purinyl, quinolinyl, isoquinolinyl, quinoxalinyl, pteridinyl, cinnoline base, quinazolinyl, indolizine, etc.

In one embodiment, R in the general formula ₁ is selected from the following groups:

In some embodiments, the general formula 1 may have n R ₂ at appropriate positions, wherein n is 0, 1, 2, 3 or 4. In some embodiments, the general formula 1 may have n R ₂ at appropriate positions, wherein n is 0, 1 or 2.

In some embodiments, each R ₂ in the general formula 1 independently represents halogen, C _1-4 alkyl, or halogenated C _1-4 alkyl.

In some embodiments, each R ₂ in the general formula 1 independently represents F, Cl, methyl, ethyl, CF ₃ , CH ₂ F, CHF ₂ , CCl ₃ , CH ₂ Cl or CHCl ₂ .

In some embodiments, each R ₂ in the general formula 1 independently represents F.

In some embodiments, in the general formula 1, X is CH or N; Y is NH; R ₁ represents optionally substituted by one or two substituents selected from halogen or C _1-4 alkyl 8-10 membered bicyclic heteroaryl containing 1 to 3 heteroatoms selected from N, O and S; _each R independently represents halogen or C _1-4 alkyl; and n is 0 , 1 or 2.

In some embodiments, in the general formula 1, X is CH or N; Y is NH; R ₁ represents optionally substituted by one or two substituents selected from halogen or C _1-4 alkyl triazolopyridyl, naphthyridyl, quinolinyl; each _R independently represents F, Cl, methyl or ethyl; and n is 0, 1 or 2.

In some embodiments, in the general formula 1, X is CH or N; Y is NH; _R is selected from:

每个R₂独立地表示F；且n为0、1或2。

each R ₂ independently represents F; and n is 0, 1 or 2.

In one embodiment, the compound of general formula 1 described in the present application is selected from:

In one embodiment, the compound of general formula 1 described in the application is not:

Compounds of general formula 1 contain asymmetric or chiral centers and, therefore, exist in different stereoisomeric forms. All stereostructures and mixtures of general formula 1, including racemic mixtures, are included as part of this application.

In one embodiment, the pharmaceutically acceptable salts of the compound of general formula 1 or its stereoisomer include but not limited to: acetate, adipate, alginate, aspartate, Benzoate, Benzenesulfonate, Bisulfate, Butyrate, Citrate, Camphorate, Camphorsulfonate, Cyclopentane Propionate, Digluconate, Lauryl Sulfate, Ethylsulfonate, Fumarate, Glucoheptanoate, Glycerophosphate, Hemisulfate, Heptanoate, Hexanoate, Hydrochloride, Hydrobromide, Hydroiodide, 2-Hydroxyethanesulfonic Acid Salt, lactate, malate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, thiocyanate, toluenesulfonate and undecanoate .

The preparation method of the compound of general formula 1, its stereoisomer or its pharmaceutically acceptable salt

The application provides the general synthetic route of the compound of synthetic general formula 1, as follows:

Step 1: Reaction of acetonitrile and potassium hexamethyldisilazide in a suitable solvent such as tetrahydrofuran (THF) under suitable reaction conditions such as nitrogen protection, at a temperature ranging from -78°C to room temperature such as -70°C for an appropriate time such as After 1 hour, the compound 1 solution was slowly added dropwise, and the temperature of the system was slowly raised to room temperature for a suitable reaction time, for example, 16 hours. The crude product was purified by silica gel column chromatography to obtain compound 2.

Step 2: react compound 2 with hydrazine hydrate in a suitable solvent such as a 2:1 mixture of ethanol and water at a suitable temperature such as -80° C. for a suitable time such as overnight. Add water, extract with an organic solvent, wash the organic phase, dry, filter, and distill under reduced pressure, and perform purification on a silica gel column chromatography to obtain compound 3.

Step 3: Dissolve compound 3 in a suitable organic solvent such as N,N-dimethylformamide, in a condensing agent such as 2-(7-azobenzotriazole)-N,N,N',N '-Tetramethyluronium hexafluorophosphate (HATU), a base such as N,N-diisopropylethylamine (DIEA) is reacted with acid 4 under appropriate conditions for an appropriate time, e.g. at room temperature for reaction 1 Hour. The reaction product was added with water, extracted with an organic solvent, and the organic phase was washed, dried, filtered, and distilled under reduced pressure, and purified by silica gel column chromatography to obtain compound 5.

Step 4: deprotect compound 5 to obtain compound 6, for example, the deprotection can be carried out at room temperature under acidic conditions such as trifluoroacetic acid.

Alternatively, the following preparation methods can also be taken from compound 3 to compound 6:

Step 5: Protect the 1-position N on the pyrazole ring in compound 3 to obtain compound 3a. For example, the protecting group in compound 3a can be (trimethylsilyl)ethoxymethyl (SEM), which can be obtained by reacting with 2- (Trimethylsilyl)ethoxymethyl chloride reacts under appropriate conditions.

Step 6: Reaction of acid compound 4 with oxalyl chloride under appropriate conditions to form acid chloride compound 4a.

Step 7: react compound 3a with acid chloride compound 4a in the presence of a base such as pyridine under appropriate conditions such as ice bath conditions for a suitable time such as 1 hour. The reaction product was added with ice water, extracted with an organic solvent, and the organic phase was washed, dried, filtered, and distilled under reduced pressure, and then purified by preparative chromatography to obtain compound 5a.

Step 8: deprotect compound 5a to obtain compound 6, for example, the deprotection can be carried out at room temperature under acidic conditions such as hydrogen chloride in 1,4-dioxane solution.

Step 9: Dissolve compound 6 in a suitable organic solvent such as N,N-dimethylformamide, in a condensing agent such as 2-(7-azobenzotriazole)-N,N,N',N '-Tetramethyluronium hexafluorophosphate (HATU), the presence of a base such as N,N-diisopropylethylamine (DIEA) and the acid compound 7 under appropriate conditions for an appropriate time, e.g. at room temperature 1 hour. The reaction product is added with water, extracted with an organic solvent, and the organic phase is washed, dried, filtered, and distilled under reduced pressure, and then purified by preparative HPLC chromatography to obtain a compound of general formula 1.

The stereoisomers of the compound of the general formula 1 of the present invention can be obtained by: using an optically active raw material to react under conditions that do not cause racemization or epimerization; or making an appropriate raw material with " chiral auxiliaries" which can subsequently be removed at an appropriate stage by derivatization; or the obtained racemic or other mixtures, based on their different physicochemical properties, by well-known means, such as , fractional crystallization or HPLC techniques for separation. Enantiomers can also be obtained by separation using chiral chromatographic columns.

The pharmaceutically acceptable salt of the compound of the general formula 1 or its stereoisomer in the present application can be formed by making the N atom on the compound or its stereoisomer structure with inorganic acid or organic acid by methods known in the art. The inorganic acid includes, but is not limited to: hydrochloric acid, hydrobromic acid, hemisulfuric acid, sulfuric acid, hydroiodic acid, and the like. The organic acids include, but are not limited to: acetic acid, adipic acid, alginic acid, aspartic acid, benzoic acid, benzenesulfonic acid, butyric acid, citric acid, camphoric acid, camphorsulfonic acid, cyclopentanepropionic acid, Gluconic acid, lauryl sulfate, ethanesulfonic acid, fumaric acid, glucoheptanoic acid, glycerophosphoric acid, heptanoic acid, caproic acid, 2-hydroxyethanesulfonic acid, lactic acid, malic acid, maleic acid, methanesulfonic acid, 2 -Naphthalenesulfonic acid, nicotinic acid, oxalic acid, thiocyanic acid, toluenesulfonic acid and undecanoic acid.

pharmaceutical composition

The present application also provides a pharmaceutical composition comprising a therapeutically effective amount of the compound of general formula 1 as defined above, its stereoisomer or a pharmaceutically acceptable salt thereof, optionally one or more additional Therapeutic or preventive agents, and pharmaceutically acceptable carriers.

In some embodiments, the compound of formula 1 is selected from:

In one embodiment, the pharmaceutically acceptable salts of the compound of general formula 1 or its stereoisomer include but not limited to: acetate, adipate, alginate, aspartate, Benzoate, Benzenesulfonate, Bisulfate, Butyrate, Citrate, Camphorate, Camphorsulfonate, Cyclopentane Propionate, Digluconate, Lauryl Sulfate, Ethylsulfonate, Fumarate, Glucoheptanoate, Glycerophosphate, Hemisulfate, Heptanoate, Hexanoate, Hydrochloride, Hydrobromide, Hydroiodide, 2-Hydroxyethanesulfonic Acid Salt, lactate, malate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, thiocyanate, toluenesulfonate and undecanoate .

In one embodiment, the additional therapeutic or prophylactic agent may be any one or more additional therapeutic or prophylactic agents as described below in the "Additional therapeutic or prophylactic agent" section.

In one embodiment, the additional therapeutic or prophylactic agent may be selected from at least one of interferon, pegylated interferon, or nucleoside analogs.

In one embodiment, the nucleoside analogue is selected from entecavir, tenofovir disoproxil fumarate and tenofovir alafenamide.

In one embodiment, the application provides a pharmaceutical composition, which comprises the compound of formula 1 described in the application (such as compound CP9, CP15, CP38 or any combination thereof), its stereoisomer Or pharmaceutically acceptable salts and nucleoside analogs thereof, and pharmaceutically acceptable carriers.

In one embodiment, the application provides a pharmaceutical composition, which comprises the compound of formula 1 described in the application (such as compound CP9, CP15, CP38 or any combination thereof), its stereoisomer Or its pharmaceutically acceptable salt and nucleoside analog, and pharmaceutically acceptable carrier, said nucleoside analog is selected from entecavir, tenofovir disoproxil fumarate and tenofovir aira Phenylamines.

In the pharmaceutical composition of the present application, the compound of the general formula 1, its stereoisomer or its pharmaceutically acceptable salt and another therapeutic agent or preventive agent can be made into one dosage form, or can be prepared separately Separate dosage forms, administered sequentially or simultaneously as a combination product.

In one embodiment, the pharmaceutical composition may be administered by a route selected from oral, rectal, nasal, pulmonary, topical, buccal and sublingual, vaginal, parenteral, subcutaneous, intramuscular, intravenous, Intradermal, intrathecal and epidural. In one embodiment, the pharmaceutical composition is administered orally. In one embodiment, the pharmaceutical composition is administered by intravenous injection.

In one embodiment, the pharmaceutical composition is an oral formulation. In one embodiment, the pharmaceutical composition is in the form of a tablet or capsule.

In one embodiment, the pharmaceutical composition of the present application can be used to treat or prevent hepatitis B.

In one embodiment, the pharmaceutical composition of the present application can reduce hepatitis B virus (HBV) load, HBsAg level and/or HBeAg level.

In one embodiment, the pharmaceutical composition of the present application can simultaneously reduce the load of hepatitis B virus (HBV), HBsAg level and HBeAg level.

In one embodiment, the pharmaceutical composition of the present application can reduce HBsAg level and/or HBeAg level.

In one embodiment, the pharmaceutical composition of the present application can reduce HBsAg level and HBeAg level at the same time.

In one embodiment, the pharmaceutical composition of the present application can eliminate HBsAg and/or HBeAg.

In one embodiment, the pharmaceutical composition of the present application can eliminate HBsAg and HBeAg at the same time.

Therefore, the present application also provides the use of the pharmaceutical composition described in the present application in the preparation of a medicament for treating or preventing hepatitis B. In one embodiment, the present application provides the use of the pharmaceutical composition described in the present application in the preparation of a medicament for reducing hepatitis B virus (HBV) load, HBsAg level and/or HBeAg level. In one embodiment, the present application provides the use of the pharmaceutical composition described in the present application in the preparation of a medicine for simultaneously reducing the load of hepatitis B virus (HBV), HBsAg level and HBeAg level. In one embodiment, the present application provides the use of the pharmaceutical composition described in the present application in the preparation of a medicament for simultaneously reducing the HBsAg level and the HBeAg level. In one embodiment, the present invention provides the use of the pharmaceutical composition described in this application in the preparation of a medicament for eliminating HBsAg and/or HBeAg. In one embodiment, the present application provides the use of the pharmaceutical composition described in the present application in the preparation of a medicament for simultaneously clearing HBsAg and HBeAg.

The compound of general formula 1, its stereoisomer or the purposes of pharmaceutically acceptable salt thereof

The present application provides the use of the compound of general formula 1 as defined above, its stereoisomer or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating or preventing hepatitis B.

In some embodiments, the compound of formula 1 is selected from:

In one embodiment, the application provides the compound of general formula 1 as defined above, its stereoisomer or pharmaceutically acceptable salt thereof in preparation for reducing hepatitis B virus (HBV) load, HBsAg level and/or HBeAg levels in medicine.

In one embodiment, the present application provides the compound of general formula 1 as defined above, its stereoisomer or pharmaceutically acceptable salt thereof in the preparation for simultaneously reducing hepatitis B virus (HBV) load, HBsAg Levels and uses of drugs for HBeAg levels.

In one embodiment, the application provides the use of the compound of general formula 1 as defined above, its stereoisomers or pharmaceutically acceptable salts thereof in the preparation of a medicament for reducing HBsAg level and/or HBeAg level .

In one embodiment, the present application provides the use of the compound of general formula 1 as defined above, its stereoisomer or a pharmaceutically acceptable salt thereof in the preparation of a medicament for simultaneously reducing HBsAg level and HBeAg level.

In one embodiment, the present application provides the use of the compound of general formula 1 as defined above, its stereoisomer or a pharmaceutically acceptable salt thereof in the preparation of a medicament for eliminating HBsAg and/or HBeAg.

In one embodiment, the present application provides the use of the compound of general formula 1 as defined above, its stereoisomer or a pharmaceutically acceptable salt thereof in the preparation of a medicament for simultaneously clearing HBsAg and HBeAg.

In one embodiment, the medicament further comprises one or more additional therapeutic or prophylactic agents. The one or more additional therapeutic or prophylactic agents may be any one or more additional therapeutic or prophylactic agents as described below in the "Additional therapeutic or prophylactic agents" section.

In one embodiment, the additional therapeutic or prophylactic agent is selected from at least one of interferon, pegylated interferon, or nucleoside analogs.

In one embodiment, the nucleoside analogue is selected from entecavir, tenofovir disoproxil fumarate and tenofovir alafenamide.

In one embodiment, the medicament is administered by a route selected from the group consisting of oral, rectal, nasal, pulmonary, topical, buccal and sublingual, vaginal, parenteral, subcutaneous, intramuscular, intravenous, intradermal, Intrathecal and epidural. In one embodiment, the medicament is administered orally. In one embodiment, the medicament is administered by intravenous injection.

In one embodiment, the medicament is an oral formulation. In one embodiment, the medicament is in the form of a tablet or capsule.

In the medicine of the present application, the compound of the general formula 1, its stereoisomer or its pharmaceutically acceptable salt and another therapeutic agent or preventive agent can be made into a dosage form, or can be made into independent Dosage forms for sequential or simultaneous administration as a combination product.

Hepatitis B

Hepatitis B is an infectious disease mainly caused by hepatitis B virus with liver lesions. Clinically, the main manifestations are loss of appetite, nausea, upper abdominal discomfort, pain in the liver area, and fatigue. Some patients may have jaundice, fever and hepatomegaly with liver function damage. Some patients can become chronic and even develop into liver cirrhosis, and a few can develop into liver cancer.

The pathogenic factor of hepatitis B virus is hepatitis B virus, abbreviated as HBV, and hepatitis B virus is a DNA virus. The genome is double-stranded, circular, partially closed DNA. The outermost layer of the virus is the outer membrane or coat of the virus, and the inner layer is the core part. The nucleoprotein is the core antigen (HBcAg), which cannot be detected in serum. In the serum of HBsAg-positive patients, three types of particles can be seen under the electron microscope: round particles with a diameter of 22 nm and filamentous particles, and less spherical particles with a diameter of 42 angstroms, also known as Dane’s particles, which are complete HBV particles.

The signs of hepatitis B are detected as follows: ① HBsAg and anti-HBs: HBsAg positive indicates that HBV is currently in the infection stage, anti-HBs is immune protective antibody positive indicates that immunity to HBV has been produced. Chronic HBsAg carriers are diagnosed on the basis of no clinical symptoms and signs, normal liver function, and HBsAg positive for more than 6 months. ②HBeAg and anti-HBe: HBeAg positive is an indicator of active HBV replication and strong infectivity. The change of the tested serum from HBeAg positive to anti-HBe positive indicates that the disease has been relieved and the infectivity has weakened. ③HBcAg and anti-HBc: Positive HBcAg indicates the existence of complete HBV particle direct reaction, and the active replication of HBV is rarely used in clinical practice due to the complexity of detection methods. Anti-HBc is a sign of HBV infection, and anti-HBc IgM positive indicates that it is in the early stage of infection and there is virus replication in the body. In chronic mild hepatitis B and HBsAg carriers, HBsAg, HBeAg and anti-HBc are all positive and highly contagious indicators are difficult to turn negative.

Additional therapeutic or prophylactic agents

In one embodiment, the compound of general formula 1 described in the present application, its stereoisomer or pharmaceutically acceptable drug or pharmaceutical composition thereof may also contain one or more additional therapeutic or prophylactic agents .

In one embodiment, the additional therapeutic or prophylactic agent may be selected from at least one of interferon, pegylated interferon, or nucleoside analogs.

In one embodiment, the additional therapeutic or prophylactic agent is selected from interferons or nucleoside analogs.

In one embodiment, the additional therapeutic or prophylactic agent is selected from nucleoside analogs.

In one embodiment, the nucleoside analogue is selected from entecavir, tenofovir disoproxil fumarate and tenofovir alafenamide.

In some embodiments, the additional therapeutic agent or preventive agent is selected from one or more of entecavir, tenofovir disoproxil fumarate and tenofovir alafenamide, for example selected from One of entecavir, tenofovir disoproxil fumarate and tenofovir alafenamide or selected from entecavir, tenofovir disoproxil fumarate and tenofovir alafenamide at least two of the amines.

The chemical name of Entecavir (Entecavir, ETV) is 2-amino-1,9-dihydro-9-[(1S,3R,4S)-4-hydroxy-3-(hydroxymethyl)-2-methylene ring Pentane]-6H-purin-6-one, its structural formula is as follows:

US Patent US5206244A discloses entecavir and its use for treating hepatitis B virus; WO9809964A1 discloses a new entecavir synthesis method; WO0164421A1 discloses a low-dose entecavir solid preparation.

Entecavir is a highly effective antiviral agent developed by Bristol-Myers Squibb in the 1990s and has a strong anti-HBV effect. It can become active triphosphate through phosphorylation, and the half-life of triphosphate in the cell is 15h. By competing with the natural substrate of HBV polymerase, deoxyguanosine triphosphate, entecavir triphosphate inhibits all three activities of the viral polymerase (reverse transcriptase): (1) initiation of HBV polymerase; (2) pregenomic mRNA Formation of negative strand of reverse transcription; (3) synthesis of positive strand of HBV DNA.

The chemical name of tenofovir disoproxil fumarate (TDF) is (R)-[[2-(6-amino-9H-purin-9-yl)-1-methylethoxy Base] methyl] phosphonic acid diisopropoxycarbonyl methyl ester fumarate is the ester precursor of tenofovir, which belongs to a new type of nucleotide reverse transcriptase inhibitor and has the activity of inhibiting HBV virus.

TDF is another new ring-opening phosphonic acid nucleoside compound successfully developed by Gilead in the United States after adefovir dipivoxil. It was first launched in the United States in October 2001, and is currently on the market in Europe, Australia, Canada and other countries.

TDF can inhibit viral polymerase in vivo by competitively binding to natural deoxyribose substrates, and terminate DNA chain synthesis by inserting into DNA. Its main mechanism of action is that it is hydrolyzed into tenofovir after oral administration, and tenofovir is phosphorylated by cellular kinases to generate a pharmacologically active metabolite tenofovir diphosphate, which is combined with 5'-deoxyadenosine triphosphate Competing, participating in the synthesis of viral DNA, after entering the viral DNA, due to the lack of 3'-OH group, the DNA elongation is blocked, thereby blocking the replication of the virus. Clinical application shows that TDF has significant anti-HBV virus curative effect, and has less toxic and side effects, so it has great clinical application prospects.

Tenofovir alafenamide is a prodrug of a new nucleoside reverse transcriptase inhibitor (NRTI) tenofovir developed by Gilead Sciences of the United States. Compared with the previous generation of anti-hepatitis B similar drug tenofovir disoproxil TDF, the antiviral activity of tenofovir alafenamide is 10 times, its stability in plasma is 200 times, and its half-life is shorter than that of It is improved by 225 times. Compared with TDF, tenofovir alafenamide only needs one tenth of the dose of TDF to achieve the same antiviral efficacy as TDF. Therefore, when tenofovir alafenamide is used for the prevention and/or treatment of hepatitis B virus (HBV) infection, it has better curative effect, higher safety and lower drug resistance.

Route of administration

The medicament or pharmaceutical composition of the present application may be administered by any route suitable for the condition to be treated. Suitable routes include oral, rectal, nasal, pulmonary, topical, buccal and sublingual, vaginal and parenteral, subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural. Those skilled in the art will understand that the particular route of administration may vary depending on, for example, the dosage form of the drug, the condition of the recipient.

In one embodiment, the medicament or pharmaceutical composition of the present application can be administered by intravenous injection.

One advantage of the medicaments or pharmaceutical compositions of the present application is that they are orally bioavailable and can be administered orally. Therefore, in one embodiment, the medicament or pharmaceutical composition of the present application can be administered orally. In one embodiment, the drug or pharmaceutical composition of the present application can be administered orally in the form of tablets or capsules.

Formulation and formulation of pharmaceutical compositions or medicaments

In certain embodiments, a compound of Formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof is administered in the form of a pharmaceutical composition. The pharmaceutical compositions of the present application can be formulated with conventional carriers and excipients which will be selected according to usual practice. Tablets will contain excipients, glidants, fillers, binders, and the like. Aqueous formulations are prepared in sterile form and, when intended for delivery by parenteral administration, are generally isotonic. The pharmaceutical compositions or medicaments of the present application, or all formulations thereof, will optionally contain excipients, such as those described in the "Handbook of Pharmaceutical Excipients" (1986). Excipients include ascorbic acid and other antioxidants, chelating agents such as EDTA, carbohydrates such as dextran, hydroxyalkylcellulose, hydroxyalkylmethylcellulose, stearic acid, and the like. The pH of the formulations ranges from about 3 to about 11, but usually from about 7 to 10. In some embodiments, the pH of the formulation ranges from about 2 to about 5, but usually from about 3 to 4.

Formulations include those suitable for the aforementioned routes of administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Techniques and formulations are generally found in Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton, PA). Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then shaping the product if necessary.

Formulations of the present application suitable for oral administration may be presented as discrete units, such as capsules or tablets, each containing a predetermined amount of the active ingredient; powders or granules; solutions or suspensions in aqueous or non-aqueous liquids; or aqueous Oil-in-liquid emulsion or water-in-oil liquid emulsion.

A tablet is made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as powder or granules, optionally with a binder, lubricant, inert diluent, preservative, surface active or dispersing agent. agent mix. Molded tablets may be made by molding in a suitable machine a mixture of the powdered active ingredient moistened with an inert liquid diluent. The tablets may optionally be coated or scored and optionally are formulated so as to provide slow or controlled release of the active ingredient therefrom.

Formulations for oral use may also be presented as hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent such as calcium phosphate or kaolin, or as soft gelatin capsules in which the active ingredient is mixed with an aqueous or oily medium such as peanut oil, liquid paraffin, or olive oil blend.

The pharmaceutical composition or drug of the present application can also be in the form of sterile injectable preparations, such as sterile injectable aqueous or oily suspensions. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, for example a solution in 1,3-butanediol, or prepared as a lyophilized powder. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are likewise used in the preparation of injectables. Acceptable vehicles and solvents that may be employed include water, Ringer's solution, isotonic sodium chloride solution and hypertonic sodium chloride solution.

definition

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs.

In this application, the use of the singular also includes the plural unless specifically stated otherwise. It should also be noted that the use of "or" means "and/or" unless stated otherwise.

The term "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes that said event or circumstance occurs and that it does not. For example, "optionally substituted alkyl" refers to "unsubstituted alkyl" (alkyl not substituted with a substituent) or "substituted alkyl" (alkyl substituted with a substituent) according to the definitions below .

In the present application, the term "halogen" means fluorine, chlorine, bromine or iodine, preferably fluorine or chlorine.

In the present application, the term "alkyl" means a straight or branched chain group consisting only of carbon atoms and hydrogen atoms, containing no unsaturated bonds and connected to the rest of the molecule by a single bond. The term "C _1-4 alkyl" refers to an alkyl group having 1 to 4 carbon atoms. Examples of C _1-4 alkyl include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, and the like.

In the present application, the term "halogenated C _1-4 alkyl" refers to a C _1-4 alkyl substituted by one or more halogen atoms, wherein C _1-4 alkyl is as defined above. Examples of _{haloC1-4alkyl} include, but are not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, 2-fluoroethyl, 2,2-difluoroethyl, chloromethyl, 2-chloro Ethyl, dichloromethyl, trichloromethyl, 1,2-dichloroethyl, bromomethyl, 3-bromopropyl, iodomethyl, etc.

In the present application, the term "5-10 membered heteroaryl" means 5-membered, 6-membered, 7-membered, 8-membered heteroatoms with 1 to 4 heteroatoms selected from nitrogen, oxygen and sulfur in the ring, and at least one aromatic ring. 1-membered, 9-membered or 10-membered ring system groups. The 5-10 membered heteroaryl can be a monocyclic or bicyclic system. The nitrogen, carbon or sulfur atom in the 5-10 membered heteroaryl can be optionally oxidized; the nitrogen atom can be optionally quaternized. Examples of 5-10 membered heteroaryl groups include, but are not limited to, thienyl, furyl, pyrrolyl, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, oxadiazolyl, Triazolyl, tetrazolyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, triazinyl, benzimidazolyl, benzopyrazolyl, benzothiazolyl, benzothienyl, imidazopyridine base, thienopyridyl, thienopyrimidinyl, thienopyrazinyl, thienopyridazinyl, thiazolopyridyl, isothiazolopyridyl, furopyrimidinyl, furopyridazinyl, triazolopyridine base, imidazopyrimidinyl, imidazopyrazinyl, imidazopyridazinyl, indolyl, indolyl, indazolyl, isoindazolyl, purinyl, quinolinyl, isoquinolyl, quinoxa Linyl, pteridinyl, cinnolinyl, quinazolinyl, indolizine, etc.

In the present application, the term "stereoisomer" refers to isomers resulting from differences in the arrangement of atoms in a molecule in space. Compounds of general formula 1 contain asymmetric or chiral centers and, therefore, exist in different stereoisomeric forms. All stereostructures and mixtures of general formula 1, including racemic mixtures, are included as part of this application. Diastereomeric mixtures can be separated into individual diastereomers on the basis of their different physicochemical properties by well known means, e.g. resolution of enantiomers can be converted into non-isomeric diastereomers by reaction with appropriate optically active substances. Enantiomers, which are separated and converted (eg hydrolyzed) to the corresponding single isomers. Enantiomers can also be separated using chiral chromatographic columns.

In the present application, the term "pharmaceutically acceptable" refers to a substance that does not affect the biological activity or properties of the compound of the present application, and is relatively non-toxic, that is, the substance can be administered to an individual without causing adverse biological reactions or adverse effects. means to interact with any component contained in the composition.

In the present application, the term "pharmaceutically acceptable salt" refers to a salt that retains the biological efficacy of the free acid and free base of the compound of the present application and has no adverse effects in biology or other aspects.

As used herein, the term "therapeutically effective amount" or "effective amount" refers to an amount that is effective at dosages and for the period of time required to achieve the desired therapeutic result. The therapeutically effective amount will depend on factors such as the nature and severity of hepatitis B or its symptoms, the particular therapeutic agent, the general condition of the recipient (e.g., height, weight, age, and physical condition), and can be determined by those skilled in the art. Determined by known standard clinical techniques.

In this application, the term "treating" may mean, for example, alleviating symptoms, prolonging survival, improving quality of life, and the like. Treatment need not be a "cure." Treatment can also refer to functional cure and clearance of HBV.

In the present application, the term "reducing the hepatitis B virus (HBV) load" refers to reducing the detectable amount of HBV DNA in the patient's blood.

In the present application, the term "reducing the level of HBsAg and/or HBeAg" refers to reducing the detectable amount of hepatitis B virus HBsAg and/or HBeAg in the patient's blood. The amount of HBsAg and/or HBeAg is usually closely related to the functional cure of hepatitis B.

Detailed ways

The following examples are for illustrative purposes only and do not limit the application. Additional objects, advantages and novel features of the present application will become apparent to those of ordinary skill in the art after reading the following examples.

Example

Embodiment 1: the preparation of compound CP15

step 1:

Tetrahydrofuran (15 mL) was added to acetonitrile (253.10 mg, 6.16 mmol, 1.5 equiv), the temperature of the system was lowered to -70 ° C, and potassium hexamethyldisilazide (0.9 g , 4.52mmol, 1.1equiv), react at -70℃ for 1 hour, compound 1: 1-(tert-butyl) 2-methyl(S)-piperidine-1,2-dicarboxylate (1g, 4.11 mmol, 1.0 equiv) was dissolved in tetrahydrofuran (5 mL), and slowly added dropwise to the reaction system, and the temperature of the system was slowly raised to room temperature for 16 hours. After adding water to terminate the reaction, the solvent was directly evaporated to dryness, and the obtained crude product was purified by silica gel column chromatography, and the ratio of eluent petroleum ether to ethyl acetate was 4/1 to obtain compound 2: (S)-2-(2-cyano Acetyl)piperidine-1-carboxylic acid tert-butyl ester (550 mg, yield: 55.04%), as a yellow oil.

Step 2:

Compound 2: (S)-tert-butyl 2-(2-cyanoacetyl)piperidine-1-carboxylate (550 mg, 2.18 mmol, 1.0 equiv) was dissolved in ethanol (4 mL) and water (2 mL), Add hydrazine hydrate (163.68mg, 3.27mmol, 1.5equiv) at room temperature, react overnight at 80°C, add water to terminate the reaction, extract the aqueous phase with ethyl acetate three times, wash the organic phase once with saturated saline, dry and filter over anhydrous sodium sulfate Afterwards, distillation under reduced pressure, the resulting crude product was purified by silica gel column chromatography, the ratio of eluent dichloromethane to methanol was 10/1, to obtain compound 3: (S)-2-(3-amino-1H-pyrazole -5-yl)piperidine-1-carboxylic acid tert-butyl ester (400 mg, yield: 68.90%, LCMS purity: 100%), as a pale yellow solid.

LCMS conditions: column: Ascentis Express C18; length: 50mm, inner diameter: 3.0mm; mobile phase A: water/0.05% trifluoroacetic acid; mobile phase B: acetonitrile/0.05% trifluoroacetic acid; time: 2.00min; Rt: 0.836 min.

ESI-MS m/z: 267.00 [M+H] ⁺ .

Step 3:

Compound 3: (S)-2-(3-Amino-1H-pyrazol-5-yl)piperidine-1-carboxylic acid tert-butyl ester (200 mg, 0.75 mmol, 1.0 equiv) was dissolved in N,N-di Methylformamide (5 mL), followed by the addition of compound 4: 4,6-difluoroindole-2-carboxylic acid (177.64 mg, 0.90 mmol, 1.2 equiv), 2-(7-azobenzotriazole )-N,N,N',N'-tetramethyluronium hexafluorophosphate (342.62mg, 0.90mmol, 1.2equiv) and N,N-diisopropylethylamine (291.15mg, 2.25mmol, 3.0equiv ). React at room temperature for 1 hour, add water to terminate the reaction, extract the aqueous phase with ethyl acetate three times, wash the organic phase with saturated brine once, dry over anhydrous sodium sulfate, filter, distill under reduced pressure, purify by silica gel column chromatography, eluent petroleum A 1/1 ratio of ether to ethyl acetate afforded compound 5: (S)-2-(3-(4,6-difluoro-1H-indole-2-carboxamido)-1H-pyrazole- 5-yl)piperidine-1-carboxylate tert-butyl ester (80 mg, yield: 23.92%, LCMS purity: 100%), as a bright yellow solid.

LCMS conditions: column: Ascentis Express C18; length: 50mm, inner diameter: 3.0mm; mobile phase A: water/0.05% trifluoroacetic acid; mobile phase B: acetonitrile/0.05% trifluoroacetic acid; time: 2.00min; Rt: 1.187 min.

ESI-MS m/z: 446.05 [M+H] ⁺ .

Step 4:

To compound 5: (S)-2-(3-(4,6-difluoro-1H-indole-2-carboxamido)-1H-pyrazol-5-yl)piperidine-1-carboxylic acid tert To a solution of the butyl ester (90 mg, 0.20 mmol, 1.0 equiv) in dichloromethane (1.6 mL) was added trifluoroacetic acid (0.4 mL). The reaction was carried out at room temperature for 1 hour, and the completion of the reaction was monitored by LCMS. The solvent was directly evaporated to dryness to obtain compound 6: (S)-4,6-difluoro-N-(5-(piperidin-2-yl)-1H-pyrazol-3-yl)-1H-indole-2 - Formamide (69 mg, yield: 98.89%, LCMS purity: 98.73%) as a bright yellow solid.

LCMS conditions: column: Ascentis Express C18; length: 50mm, inner diameter: 3.0mm; mobile phase A: water/0.05% trifluoroacetic acid; mobile phase B: acetonitrile/0.05% trifluoroacetic acid; time: 2.00min; Rt: 0.869 min.

ESI-MS m/z: 346.00 [M+H] ⁺ .

Step 5:

Compound 6: (S)-4,6-difluoro-N-(5-(piperidin-2-yl)-1H-pyrazol-3-yl)-1H-indole-2-carboxamide (69mg , 0.20mmol, 1.0equiv) was dissolved in N,N-dimethylformamide (2mL), then sequentially added compound 7: [1,2,4]triazolo[1,5-a]pyridine-6-carboxy acid (39.11mg, 0.24mmol, 1.2equiv), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (91.16mg, 0.24mmol , 1.2 equiv) and N,N-diisopropylethylamine (129.11 mg, 1.00 mmol, 5.0 equiv). React at room temperature for 1 hour, stop the reaction with water, extract 3 times with ethyl acetate, wash the organic phase once with saturated brine, dry over anhydrous sodium sulfate, filter, distill under reduced pressure, and prepare the target compound CP15 by high-pressure liquid phase: (S) -N-(5-(1-([1,2,4]triazolo[1,5-a]pyridine-6-carbonyl)piperidin-2-yl)-1H-pyrazol-3-yl) -4,6-difluoro-1H-indole-2-carboxamide (12.8 mg, yield: 13.05%, LCMS purity: 99.88%), as a white solid.

LCMS conditions: column: Luna Omega; length: 50 mm, inner diameter: 3.0 mm; mobile phase A: water/0.09% formic acid; mobile phase B: acetonitrile/0.1% formic acid; time: 2.00 min; Rt: 1.102 min.

ESI-MS m/z: 491.30 [M+H] ⁺ .

¹ H NMR (300MHz,DMSO-d ₆ )δ12.49(s,1H),12.08(s,1H),11.00(s,1H),9.21(s,1H),8.60(s,1H),7.94- 7.97(m,1H),7.78(d,J=9.2Hz,1H),7.61(s,1H),7.05–7.08(m,1H),6.92(t,J=10.3Hz,1H),6.63(s ,1H), 2.95(br,1H), 2.35-2.45(m,2H), 2.15-2.30(m,1H), 1.92(m,1H), 1.45-1.75(m,4H).

Embodiment 2: the preparation of compound CP38

step 1:

Add tetrahydrofuran (30mL) to acetonitrile (0.76g, 18.49mmol, 1.5equiv), lower the temperature of the system to -70°C, slowly add a tetrahydrofuran solution of potassium hexamethyldisilazide to the system under nitrogen protection (13.56 mL, 13.56 mmol, 1.1 equiv). -70°C for 1 hour, compound 1: 1-(tert-butyl) 2-methyl(R)-piperidine-1,2-dicarboxylate (3 g, 12.33 mmol, 1.0 equiv) was dissolved in tetrahydrofuran ( 20 mL), was slowly added dropwise into the reaction system, and the temperature of the system was slowly raised to room temperature for 16 hours. After adding water to terminate the reaction, the solvent was directly evaporated to dryness, and the obtained crude product was purified by silica gel column chromatography (eluent: the ratio of petroleum ether to ethyl acetate was 5/1) to obtain compound 2: (R)-2-(2- tert-butyl cyanoacetyl)piperidine-1-carboxylate (1.6 g, yield: 51.43%), as a yellow oil.

Step 2:

Compound 2: tert-butyl (R)-2-(2-cyanoacetyl)piperidine-1-carboxylate (1.6 g, 6.34 mmol, 1.0 equiv) was dissolved in ethanol (12 mL) and water (6 mL) , add hydrazine hydrate (0.38g, 7.61mmol, 1.2equiv) at room temperature, and react at 80°C for 7 hours. Water was added to terminate the reaction, the aqueous phase was extracted three times with ethyl acetate, the organic phase was washed once with saturated brine, dried over anhydrous sodium sulfate, filtered, and distilled under reduced pressure, and the resulting crude product was purified by silica gel column chromatography (eluent: The ratio of dichloromethane to methanol is 12/1), to obtain compound 3: (R)-2-(3-amino-1H-pyrazol-5-yl)piperidine-1-carboxylic acid tert-butyl ester (1.06g , yield: 62.76%, LCMS purity: 100%), as a yellow solid.

LCMS conditions: column: HALO; length: 30 mm, inner diameter: 3.0 mm; mobile phase A: water/0.05% trifluoroacetic acid; mobile phase B: acetonitrile/0.05% trifluoroacetic acid; time: 2.00 min; Rt: 0.894 min.

ESI-MS m/z: 267.30 [M+H] ⁺ .

Step 3:

Compound 3: (R)-2-(3-Amino-1H-pyrazol-5-yl)piperidine-1-carboxylic acid tert-butyl ester (150 mg, 0.56 mmol, 1.0 equiv) was dissolved in THF (2 mL) , under ice bath conditions, slowly added N,N-diisopropylethylamine (109.18mg, 0.85mmol, 1.5equiv), and then added 2-(trimethylsilyl)ethoxymethyl to the system Chlorine (103.28 mg, 0.62 mmol, 1.1 equiv), react overnight at room temperature. The reaction was terminated by adding ice water to terminate the reaction, the aqueous phase was extracted 3 times with ethyl acetate, the organic phase was washed once with saturated brine, dried over anhydrous sodium sulfate, filtered, and distilled under reduced pressure, and the crude product obtained was purified with a silica gel plate ( Eluent: dichloromethane to methanol in a ratio of 20/1) to give compound 4: (R)-2-(3-amino-1-((2-(trimethylsilyl)ethoxy) Methyl)-1H-pyrazol-5-yl)piperidine-1-carboxylic acid tert-butyl ester (120 mg, yield: 48.50%, LCMS purity: 88.54%) as a brown oil.

LCMS conditions: column: HALO; length: 20 mm, inner diameter: 3.0 mm; mobile phase A: water/0.05% trifluoroacetic acid; mobile phase B: acetonitrile/0.05% trifluoroacetic acid; time: 2.00 min; Rt: 0.626 min.

ESI-MS m/z: 397.25 [M+H] ⁺ .

Step 4:

Compound 8: 5-fluoroindole-2-carboxylic acid (50mg, 0.28mmol, 1.0equiv) was dissolved in dichloromethane (1mL), and oxalyl chloride (38.97mg , 0.31mmol, 1.1equiv) and N,N-dimethylformamide (0.1mL), reacted for 30 minutes. The solvent was evaporated to dryness to obtain yellow oily compound 9: 5-fluoro-1H-indole-2-carbonyl chloride (55 mg), which was directly used in the next reaction without purification.

Step 5:

Compound 4: (R)-2-(3-amino-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-5-yl)piperidine-1- Tert-butyl carboxylate (110.40 mg, 0.28 mmol, 1.0 equiv) was dissolved in dichloromethane (2 mL), and compound 9: 5-fluoro-1H-indole- 2-Carbonyl chloride (55 mg, 0.28 mmol, 1.0 equiv) in dichloromethane (0.5 mL) and pyridine (110.09 mg, 1.39 mmol, 5.0 equiv). React for 1 hour, add ice water to terminate the reaction, extract the aqueous phase with ethyl acetate three times, wash the organic phase with saturated brine once, dry over anhydrous sodium sulfate, filter, and purify the crude product obtained by distillation under reduced pressure with a preparative chromatographic plate (Eluant: The ratio of petroleum ether to ethyl acetate is 1/1), to obtain compound 5 as light yellow oil: (R)-2-(3-(5-fluoro-1H-indole-2-formyl Amino)-1-(2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-5-yl)piperidine-1-carboxylic acid tert-butyl ester (60 mg, yield: 35.20 %, LCMS purity: 91.07%).

LCMS conditions: column: Ascentis Express C18; length: 50mm, inner diameter: 3.0mm; mobile phase A: water/0.05% trifluoroacetic acid; mobile phase B: acetonitrile/0.05% trifluoroacetic acid; time: 2.00min; Rt: 1.402 min.

ESI-MS m/z: 580.10 [M+Na] ⁺ .

Step 6:

To compound 5: (R)-2-(3-(5-fluoro-1H-indole-2-carboxamido)-1-(2-(trimethylsilyl)ethoxy)methyl) -1H-pyrazol-5-yl)piperidine-1-carboxylate tert-butyl ester (50mg, 0.09mmol, 1.0equiv) was added in 1,4-dioxane solution (1.5mL) of hydrogen chloride, and reacted at room temperature for 1 Hours, LCMS monitored that the reaction was complete, and the solvent was directly evaporated to dryness to obtain white solid compound 6: (R)-5-fluoro-N-(5-(piperidin-2-yl)-1H-pyrazol-3-yl) -1H-indole-2-carboxamide (40 mg, yield: 95.41%, LCMS purity: 74.29%).

LCMS conditions: column: Kinetex EVO; length: 30 mm, inner diameter: 3.0 mm; mobile phase A: water/6.5 mM ammonium bicarbonate; mobile phase B: acetonitrile; time: 2.00 min; Rt: 0.921 min.

ESI-MS m/z: 328.00 [M+H] ⁺ .

Step 7:

Compound 6: (R)-5-fluoro-N-(5-(piperidin-2-yl)-1H-pyrazol-3-yl)-1H-indole-2-carboxamide (40mg, 0.12mmol , 1.0equiv) was dissolved in N,N-dimethylformamide (2mL), then sequentially added compound 7: 2-methyl-1,6-naphthyridine-3-carboxylic acid (22.99mg, 0.12mmol, 1.0equiv) , 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (55.75mg, 0.14mmol, 1.2equiv) and N,N-diiso Propylethylamine (78.96 mg, 0.61 mmol, 5.0 equiv). React at room temperature for 1 hour, terminate the reaction with water, extract 3 times with ethyl acetate, wash the organic phase once with saturated brine, dry over anhydrous sodium sulfate, filter, and distill under reduced pressure to obtain a crude product, which was prepared by high-pressure liquid phase to obtain the target compound CP38:( R)-5-fluoro-N-(5-(1-(2-methyl-1,6-naphthyridine-3-carbonyl)piperidin-2-yl)-1H-pyrazol-3-yl)- 1H-Indole-2-carboxamide (8.7 mg, yield: 13.36%, LCMS purity: 93.36%), as a pale yellow solid.

LCMS conditions: column: Kinetex EVO; length: 30 mm, inner diameter: 3.0 mm; mobile phase A: water/6.5 mM ammonium bicarbonate; mobile phase B: acetonitrile; time: 3.00 min; Rt: 1.482 min.

ESI-MS m/z: 498.00 [M+H] ⁺ .

¹ H NMR (400MHz,DMSO-d ₆ )δ12.54(s,1H),11.80(s,1H),10.94(s,1H),9.40(s,1H),8.80-8.35(m,2H), 7.88(d,J=5.9Hz,1H),7.52-7.49(m,2H),7.09(t,J=9.2Hz,1H),6.75-6.58(m,1H),6.04(br,1H),4.55 -5.00(m,1H),3.00-3.20(m,1H),2.73(s,3H),2.30-2.42(m,1H),2.09-2.21(m,1H),1.89-1.99(m,1H) , 1.73(br,2H), 1.53(br,2H).

Example 3: Application of HepG2.2.15 cells to evaluate the in vitro anti-HBV activity of compounds CP9, CP15 and CP38

Material

Compounds CP15 and CP38 were prepared by the methods described in Examples 1 and 2;

Compound CP9 was purchased from Tianjin WuXi Kangde New Drug Development Co., Ltd., CAT#/ID: WXCD00160257_SE0026_SE1167, molecular weight: 499.962;

Entecavir was purchased from Shanghai Titan Technology Co., Ltd. (batch number: P1214012; 99.0% purity);

Cell HepG2.2.15 was purchased from Shanghai WuXi AppTec New Drug Development Co., Ltd.;

Cell subculture medium is containing 10% fetal bovine serum (FBS, ExCell product number FSP500), 500μg/ml G418, 1% glutamine, 1% NEAA (non-essential amino acid), 1mM sodium pyruvate, 1% penicillin-streptomycin DMEM medium (Gibco, Cat. No. 11960051), mainly used for subculture of cells. Cell plating medium contains 2% fetal bovine serum (FBS, ExCell product number FSP500), 500μg/ml G418, 1% glutamine, 1% NEAA (non-essential amino acid), 1mM sodium pyruvate, 1% penicillin-streptomycin DMEM culture medium (Gibco, product number 11960051), mainly used for cell plating and drug addition and replacement;

QIAamp 96DNA Blood Kit was purchased from Qiagen (Cat. No. 51162);

HBsAg ELISA kit was purchased from Antu Biology (Product No. CL0310);

HBeAg ELISA kit was purchased from Antu Biology (Product No. CL0312);

CellTiter-Glo kit was purchased from Roche;

HBV virus was purchased from Shanghai WuXi PharmaTech New Drug Development Co., Ltd.

Plating Cells and Compound Treatment

On day 0, HepG2.2.15 in cell plating medium was plated into 96-well plates (6×10 ⁴ cells/well).

On day 1, medium containing compound was added. The test compounds CP9, CP15 and CP38 were diluted with DMSO to 3 single-drug concentrations, and the reference compound ETV was diluted to 7 concentrations, 3-fold dilutions, in triplicate wells (Table 1). At the same time, a DMSO control containing only DMSO and no compound was also set up. Cells were cultured for 3 days under the condition of 5% CO ₂ and 37°C.

On day 4, replace once with fresh cell plating medium containing compound.

On the seventh day, the supernatant was collected, and the collected cell supernatant was detected by ELISA method to detect HBeAg and HBsAg, and the qPCR method was used to detect the level of HBV DNA. At the same time, CellTiter-Glo detects cell viability, and collects cells for cryopreservation (for standby).

Table 1. Compound Test Concentrations

Sample testing

Detection of HBV DNA content in cell culture supernatant by qPCR

According to the QIAamp 96DNA Blood Kit instructions, extract the DNA in the cell culture supernatant. The content of HBV DNA was detected by qPCR method. PCR reaction: 95°C, 10min; 95°C, 15sec, 60°C, 1min, 40 cycles.

Detection of HBeAg and HBsAg in Cell Culture Supernatant by ELISA

The method refers to the kit instruction manual, and the brief description is as follows: take 50 μl of standard, sample and control substance and add them to the detection plate, then add 50 μl of enzyme conjugate to each well, incubate at 37°C for 60 minutes, wash the plate with washing solution and blot dry , then add 50 μl premixed luminescence substrate, incubate at room temperature in the dark for 10 minutes, and finally measure the luminescence value with a microplate reader.

CellTiter-Glo Cell Viability Assay

The cell viability was determined according to the instructions of the CellTiter-Glo kit. The method is briefly described as follows: After collecting the cell culture supernatant, add CellTiter-Glo (1:1 dilution in medium) to each well, incubate at room temperature for 10 minutes, and measure the luminescence value with a microplate reader.

data calculation

HBV DNA inhibition rate (%)=(1-the HBV copy number of the compound group sample/the HBV copy number of the DMSO control group)×100%

HBsAg inhibition rate (%)=(1-HBsAg value of sample/HBsAg value of DMSO control group)×100%

HBeAg inhibition rate (%)=(1-HBeAg value of sample/HBeAg value of DMSO control group)×100%

Cell viability (%) = ((sample value - average value of medium control group) / (average value of DMSO group - average value of medium control group) × 100%

_EC50 values were calculated using GraphPad Prism software (four parameter logistic equations).

data analysis

The results of the reference compound ETV are shown in Table 2 below:

Table 2

The results for the control compound ETV show that the test method is valid. In addition, according to Table 2, ETV has a significant inhibitory effect on HBVDNA, but has no significant inhibitory effect on HBeAg and HBsAg.

The results of the tested compounds are shown in Table 3 below:

table 3

As shown in Table 3 and Figure 1, the cell viability of the compound CP15 of the present application is even higher than 100% at the concentration of 1 μM and 10 μM, and the cell viability is as high as 80.3% even at the concentration of 20 μM, indicating that the compound of the present application has a high Cell viability, with the potential of less toxic side effects and high safety.

As shown in Table 3 and Figure 2, the inhibitory rates of 10 μM and 20 μM CP15 on HBV DNA in HepG2-NTCP cells were 16.6% and 47.3%, respectively, indicating that the compound of the present application has a certain inhibitory effect on HBV DNA.

As shown in Table 3 and Figure 3, 10 μ M of CP15 has an inhibitory rate of 37.4% to HBeAg in HepG2.2.15 cells, while 20 μ M of CP15 can reach 70.2% of the inhibitory rate of HBeAg, showing that the compound of the present application has a significant effect on HBeAg The inhibitory effect can effectively inhibit HBeAg.

As shown in Table 3 and Figure 4, 1μM and 10μM of CP15 had an inhibitory rate of 36.7% and 51.7% on HBsAg in HepG2.2.15 cells, respectively, while 20μM of CP15 had an inhibitory rate of 92.6% on HBsAg, indicating that the present application The compound has obvious inhibitory effect on HBsAg, can effectively inhibit HBsAg, and can even completely eliminate HBsAg.

The above experimental results show that the compound of the present application has strong cell viability and low cytotoxicity, and can effectively reduce HBV viral load, HBsAg and HBeAg levels. It can be seen that the compound of the present application can effectively reduce the levels of HBeAg and HBsAg at the same time, especially the level of HBsAg, so that it is expected to eliminate hepatitis B virus, achieve functional cure, and avoid long-term or life-long medication. At the same time, it also lays a solid foundation for the combination of the compound of the application and the hepatitis B drugs in the prior art that can reduce the titer of HBV but cannot reduce the levels of HBsAg and HBeAg, so as to treat hepatitis B from different aspects.

Although the present application has been described with reference to specific embodiments, those skilled in the art will recognize that changes or improvements may be made to the described embodiments without departing from the spirit and scope of the present application, which is described in the appended The claims are limited.

Claims (10)

1. A compound or a stereoisomer or a pharmaceutically acceptable salt thereof, the compound having a structure represented by formula 1:

wherein:

x is CH or N;

y is O, S or NH;

R ₁ optionally one or more selected from halogen, C _1-4 Alkyl, halogenated C _1-4 A 5-10 membered heteroaryl substituted with a substituent of an alkyl group, the heteroaryl comprising 1 to 3 heteroatoms selected from N, O and S;

each R ₂ Independently represent halogen, C _1-4 Alkyl, halogenated C _1-4 An alkyl group; and is also provided with

n is an integer of 0 to 4,

provided that the compound is not:

2. the compound of claim 1, wherein Y is NH.

3. The compound of claim 1, wherein R ₁ Optionally one or more selected from halogen, C _1-4 Alkyl, halogenated C _1-4 An 8-10 membered bicyclic heteroaryl substituted with a substituent of alkyl, said heteroaryl comprising 1 to 3 heteroatoms selected from N, O and S.

4. The compound of claim 1, wherein R ₁ Selected from:

5. a compound according to any one of claims 1-4, selected from:

6. a pharmaceutical composition comprising a compound having a structure represented by formula 1 or a stereoisomer thereof or a pharmaceutically acceptable salt thereof; optionally one or more additional therapeutic or prophylactic agents; a pharmaceutically acceptable carrier:

wherein:

x is CH or N;

y is O, S or NH;

R ₁ optionally one or more selected from halogen, C _1-4 Alkyl, halogenated C _1-4 A 5-10 membered heteroaryl substituted with a substituent of an alkyl group, the heteroaryl comprising 1 to 3 heteroatoms selected from N, O and S;

Each R ₂ Independently represent halogen, C _1-4 Alkyl, halogenated C _1-4 An alkyl group; and is also provided with

n is an integer from 0 to 4.

7. The pharmaceutical composition of claim 6, wherein the compound is selected from the group consisting of:

8. the pharmaceutical composition according to claim 6 or 7, wherein the additional therapeutic or prophylactic agent is selected from at least one of interferon, pegylated interferon or a nucleoside analogue, and preferably wherein the nucleoside analogue is selected from entecavir, tenofovir disoproxil fumarate and tenofovir alafenamide.

9. Use of a compound having the structure shown in formula 1 or a stereoisomer or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of hepatitis b:

wherein:

x is CH or N;

y is O, S or NH;

R ₁ optionally one or more selected from halogen, C _1-4 Alkyl, halogenated C _1-4 A 5-10 membered heteroaryl substituted with a substituent of an alkyl group, the heteroaryl comprising 1 to 3 heteroatoms selected from N, O and S;

each R ₂ Independently represent halogen, C _1-4 Alkyl, halogenated C _1-4 An alkyl group; and is also provided with

n is an integer from 0 to 4.

10. The use according to claim 9, wherein the compound is selected from the group consisting of:

Images