CN111253391A - Deuterium-containing azacyclic dione compound for treating influenza - Google Patents

Abstract

The invention provides a deuterium-containing azacyclic dione compound, which is characterized in that: a compound represented by the following structural formula or a pharmaceutically acceptable salt thereof;

the compounds have proliferation inhibiting activity on influenza virus, especially on influenza-related cap-dependent endonuclease, so as to inhibit influenza virus proliferation and further treat influenza.

Description

Deuterium-containing azacyclic dione compound for treating influenza

Technical Field

The invention relates to the field of organic synthesis, in particular to a deuterium-containing azacyclic dione compound for treating influenza and a preparation method thereof.

Background

Influenza viruses belong to the Orthomyxoviridae (Orthomyxoviridae) family of viruses, which are negative-sense single-stranded RNA viruses. Three types of influenza viruses are known: influenza virus a, influenza virus B, influenza virus C. Infection with influenza virus causes acute respiratory infections and can cause other respiratory viral infections, including upper and lower respiratory viral infections. Viral infections of the upper respiratory tract involve the nose, sinuses, pharynx and/or larynx. Viral infections of the lower respiratory tract involve the respiratory system below the vocal cords, including the trachea, main bronchi and lungs. Infection with influenza virus is a leading cause of death in millions of people each year. The traditional Chinese medicine composition is a particularly important disease in high-risk groups such as infants, old people and the like, and the complication rate of pneumonia is high in the old people, and the old people account for most of death caused by influenza.

Influenza infection in humans is caused by influenza a and influenza B. Influenza A infection accounts for the majority of influenza, and the disease condition of patients is more serious. Influenza a virus has a host cell-derived lipid membrane that contains hemagglutinin, neuraminidase, and M2 proteins protruding from the surface of the virus. Influenza a viruses can be further classified according to the hemagglutinin (H or HA) and neuraminidase (N) antigen types. 16H antigens (H1 to H16) and 9N antigens (NI to N9) are now known. Influenza a viruses are divided into several subtypes based on the binding of H and N antigens. Subtypes that have now been found to exist include H1N1, H1N2, H2N2, H3N1, H3N2, H3N8, H5N1, H5N2, H5N3, H5N8, H5N9, H7N1, H7N2, H7N3, H7N4, H7N7, H7N9, H9N2 and H10N 7. Influenza polymerase is a heterotrimer composed of 3 subunits: polymerase Acid (PA), polymerase base 1(PB1), and polymerase base 2(PB 2). In the nucleus of infected cells, influenza virus polymerase is responsible for the replication and transcription of viral RNA. The PA subunit comprises an endonuclease active site. Endonuclease cleavage of host cell mRNA by PA followed by cellular mRNA used as a primer for viral mRNA synthesis by the PBl subunit.

Influenza viruses can be transmitted from person to person by direct contact with secretions of influenza patients, or with contaminated surfaces or objects. Complications of influenza virus infection include pneumonia, bronchitis, dehydration, and sinus and ear infections.

Vaccination is the primary route to control the spread of influenza viruses. However, there are limitations to the preparation of influenza vaccines, such as a6 month lag in the identification of new outbreaks of virus to the clinical use of the vaccine. Therefore, antiviral drugs are currently an urgent need for methods for the prevention and treatment of influenza.

Anti-influenza drugs that have been approved fall into two broad categories: (1) the drugs for inhibiting the uncoating process of viruses include, but are not limited to, famethrel (Symmetrel, trade name: Amantadine, and Rimantadine hydrochloride (Flumadine, trade name: Rimantadine)), and (2) neuraminidase inhibitors which inhibit the budding/release of viruses from cells, Oseltamivir (Oseltamivir, trade name: Tamiflu, Zanamivir (Zanamivir, trade name: Relenza), peramivir (permivir), and laninamivir octanoate (laninamivir octanoate).

Despite the excellent efficacy of these existing drugs, there are still a number of problems to be overcome in treating influenza, such as: the use of existing drugs has led to the emergence of resistant influenza virus strains; these drugs have considerable toxic side effects; a worldwide pandemic of new influenza viruses with high pathogenicity or lethality may occur. Therefore, there is still a need and urgency to develop new mechanisms of anti-influenza drugs.

The cap-dependent endonuclease is an enzyme derived from influenza virus, plays a key role in virus proliferation, and has virus-specific enzyme activity which is not possessed by a host, so that the cap-dependent endonuclease is a unique good target for developing a novel anti-influenza drug. The cap-dependent endonuclease activity is: a fragment containing 9 to 13 bases of a cap structure (bases of the cap structure are not included in this number) is produced using a host mRNA precursor as a substrate. This fragment functions as a primer for viral RNA polymerase and is used for the synthesis of mRNA encoding viral proteins. That is, the substance which inhibits cap-dependent endonuclease inhibits synthesis of viral protein by inhibiting synthesis of viral mRNA, thereby inhibiting viral growth.

At present, a medicament or a composition thereof which is clinically used as an anti-influenza medicament is lacking.

Disclosure of Invention

The present invention aims to provide a novel deuterium-containing azacyclic dione compound that can be used for treating influenza.

The invention provides a deuterium-containing azacyclic dione compound, which is characterized in that: a compound represented by the following structural formula or a pharmaceutically acceptable salt thereof;

wherein-O-R¹The group is selected from hydroxyl or a product obtained after the hydroxyl is reacted; the product after the hydroxyl group is reacted refers to a product obtained after the conventional substances such as acyl halide compounds, halides, sulfonyl compounds, acids, alcohols and the like are used as raw materials and generate with the hydroxyl group, and the product can exist in the forms of ethers, esters and the like.

In general, the R is¹The role of the various options as protecting groups for hydroxyl groups, such as: acetyl (Ac), 2-Methoxyethoxymethyl Ether (MEM), methoxymethyl ether (MOM), p-methoxybenzyl ether (PMB), methylthiomethyl ether (MTM), pivaloyl (Piv), Tetrahydropyran (THP), silyl ether protecting groups, methyl ether and the like, or groups which are large in the equivalent volume of sulfonyl and benzyl and are easily removed.

R²And R³Identical or different, selected from hydrogen, alkyl, substituted alkyl;

R⁴selected from hydrogen, halogen, cyano, alkyl, substituted alkyl;

X¹,X²,X³,X⁴,X⁵,X⁶,X⁷,X⁸identical or different, selected from hydrogen, deuterium, halogen, cyano, nitro, amino;

Y¹,Y²,Y³identical or different, selected from hydrogen, deuterium;

x is above¹,X²,X³,X⁴,X⁵,X⁶,X⁷,X⁸，Y¹,Y²,Y³At least one of which is deuterium.

In addition, the invention also provides a deuterium-containing azacyclic dione compound, which is characterized in that: a compound represented by the following structural formula or a pharmaceutically acceptable salt thereof;

wherein the content of the first and second substances,

r1 is selected from: hydrogen, -C (═ O) R^A,-C(＝O)OR^A,-CH₂OC(＝O)R^A,-CH₂OC(＝O)OR^A,-CH(Me)OC(＝O)R^A, -CH(Me)OC(＝O)OR^A；

R^ASelected from alkyl, substituted alkyl

R⁴Selected from hydrogen, halogen, cyano, alkyl, substituted alkyl;

n is a natural number of 0 to 5; that is, the substituents herein are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl; in addition, any one or more carbons of the cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl can be replaced by oxygen, sulfur and nitrogen to form heterocycloalkyl;

R₂' refers to any hydrogen atom on a cycloalkyl or heterocycloalkyl group being substituted with hydrogen, halogen, alkyl, substituted alkyl;

X¹,X²,X³,X⁴,X⁵,X⁶,X⁷,X⁸identical or different, selected from hydrogen, deuterium, halogen, cyano, nitro, amino;

Y¹,Y²,Y³identical or different, selected from hydrogen, deuterium;

x is above¹,X²,X³,X⁴,X⁵,X⁶,X⁷,X⁸，Y¹,Y²,Y³At least one of which is deuterium.

Furthermore, the deuterium-containing azacyclic dione compound provided by the invention also has the following characteristics: namely, the above R¹Selected from H, -C (═ O) R^A,-C(＝O)OR^A,-CH₂OC(＝O)R^A,-CH₂OC(＝O)OR^A,-CH(Me)OC(＝O)R^A, -CH(Me)OC(＝O)OR^A；

Wherein R is^ASelected from hydrogen and alkyl.

Furthermore, the deuterium-containing azacyclic dione compound provided by the invention also has the following characteristics: that is, the substituted alkyl group means that one or more hydrogen atoms in the alkyl group are substituted with a hydroxyl group, a halogen group, a cyano group, a nitro group, an amino group, an ether group, or an ester group.

In the present invention, an alkyl group generally means an alkyl group having not more than 10 carbon atoms.

Furthermore, the invention also provides a preparation method of the deuterium-containing azacyclic dione compound, which is characterized by comprising the following steps: the method comprises the following steps of (1): 1-5, carrying out substitution reaction on a substrate A and a substrate B under an acidic reaction condition to obtain a target deuterium-containing azacyclic diketone compound;

wherein the substrate A is a compound shown in the following structure:

substrate B is a compound represented by the following structure:

wherein when R is^1AIn the case of hydroxyl protecting groups, the target deuterium-containing azacyclic dione compound may also be deprotected to yield R^1AA product which is hydrogen;

when R is^1AIn the case of hydrogen, any reaction wherein hydrogen is substituted by a substituent may also occur at the hydroxyl group on the subject deuterium-containing azacyclic dione compound. Such as: ether formation, esterification and the like.

Furthermore, the preparation method of the nitrogen heterocyclic diketone compound containing deuterium provided by the invention also has the following characteristics: namely, the target deuterium-containing azacyclic dione compound is subjected to a chiral separation process to obtain a chiral product.

Furthermore, the preparation method of the nitrogen heterocyclic diketone compound containing deuterium provided by the invention also has the following characteristics: namely, the substrate A is prepared by the following steps: taking a derivative A0 of pyran formaldehyde as a raw material, and sequentially obtaining intermediate products A1, A2, A3, A4, A5, A6, A7, A8, A9 and A10 through reaction and deprotection of rear amine groups to obtain a substrate A;

wherein, the structures of the derivative A0 of the pyran formaldehyde and the intermediate products A1, A2, A3, A4, A5, A6, A7, A8, A9 and A10 are respectively as follows:

the derivatives of pyran carboxaldehyde A0 are

Intermediate A1 is

Intermediate A2 is

Intermediate A3 is

Intermediate A4 is

Intermediate A5 is

Intermediate A6 is

Intermediate A7 is

Intermediate A8 is

Intermediate A9 is

Intermediate A10 is

Wherein pro is a hydroxy protecting group;

pro' is an amine protecting group; such as: groups that can be used to protect the hydrogen on NH, such as benzyloxycarbonyl (Cbz), t-Butoxycarbonyl (BOC), 9-Fluorenylmethyloxycarbonyl (FMOC), benzyl (Bn), p-methoxyphenyl (PMP), trityl derivatives, and the like.

lea is a leaving group. Such as: alkyl, benzyl, and the like.

The specific reaction equation is as follows:

furthermore, the preparation method of the nitrogen heterocyclic diketone compound containing deuterium provided by the invention also has the following characteristics: namely, the substrate A is prepared by the following steps: taking a derivative A0 of pyran formaldehyde as a raw material, and sequentially obtaining intermediate products A1-2, A2-2, A3-2, A4-2, A5-2, A6-2 and A7-2 after amino deprotection to obtain a substrate A;

wherein, the structures of the derivative A0 of the pyran formaldehyde and the intermediate products A1-2, A2-2, A3-2, A4-2, A5-2, A6-2 and A7-2 are respectively as follows:

the derivatives of pyran carboxaldehyde A0 are

Intermediate A1-2 is

Intermediate A2-2 is

Intermediate A3-2 is

Intermediate A4-2 is

Intermediate A5-2 is

Intermediate A6-2 is

Intermediate A7-2 is

Wherein pro is a hydroxy protecting group;

pro' is an amine protecting group;

the corresponding carbon is R²，R³Substituted by a group, or by cycloalkyl, substituted cycloalkyl, heterocycloalkyl, or substituted heterocycloalkyl.

The specific reaction equation is as follows:

furthermore, the preparation method of the nitrogen heterocyclic diketone compound containing deuterium provided by the invention also has the following characteristics: namely, the substrate B is prepared by the following steps: halogenated benzene derivative B0 is used as a raw material, intermediate products B1, B2, B3 and B4 are obtained in sequence through reaction, and carbonyl is reduced into hydroxyl to obtain a substrate B;

wherein, the structures of the halogenated benzene derivative B0 and the intermediate products B1, B2, B3 and B4 are respectively shown as follows:

halogenobenzene derivative B0 is

Intermediate B1 is

Intermediate B2 is

Intermediate B3 is

Intermediate B4 is

Or

The intermediate product B4 can obtain an intermediate product B4-2 under the action of a deutero reagent, and then the intermediate product B is reduced into hydroxyl through carbonyl to obtain a substrate B;

the intermediate product B4-2 is a compound represented by the following structure:

the specific reaction equation is as follows:

in addition, the invention provides a deuterium-containing azacyclic dione compound, which is characterized in that: is used as one of the components of a medicine or a medicine composition for treating influenza.

The invention has the following functions and effects:

some embodiments disclosed herein relate to methods of ameliorating and/or treating an influenza viral infection, which may include administering to a subject infected with an influenza virus an effective amount of one or more compounds of formula (la) and prodrugs thereof, or pharmaceutically acceptable salts thereof, or a pharmaceutical composition comprising one or more compounds of formula (lb) or pharmaceutically acceptable salts thereof.

Some embodiments disclosed herein relate to methods of preventing an orthomyxovirus infection that can include administering to a subject likely to be infected with an influenza virus an effective amount of one or more compounds of formula (la) and prodrugs thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising one or more compounds of formula (lb) or a pharmaceutically acceptable salt thereof. Influenza virus infection may be caused by a subtype of influenza virus, such as influenza A, B and/or C.

It was found that carbon-deuterium bonds are slightly shorter and higher in bond energy than carbon-hydrogen bonds, thus leading to the so-called isotopic effect, i.e. carbon-deuterium bonds (C-D) are more stable than carbon-hydrogen bonds (C-H), with a first order kinetic isotopic effect of about 6.5 times (kH/kD ═ 6.5) at room temperature. The replacement of the hydrogen atom of a small molecule by deuterium can significantly improve the in vivo metabolic profile of the compound. Of course, deuterium is substituted for hydrogen at the potential metabolic sites of the drug, and such deuteration can improve the biological stability and therapeutic effect of the drug. Therefore, improving the metabolic stability of a drug by deuteration to achieve the purpose of improving the in vivo kinetics of a drug (increasing in vivo concentration, prolonging half-life, and alleviating side effects, etc.) has become an integral part of modern pharmaceutical chemistry.

Drawings

FIG. 1, pharmacokinetic results chart;

FIG. 2 is a graph showing the results of pharmacokinetics.

Detailed Description

Intermediate series 1

Intermediate A5- (benzyloxy) -2, 3-dihydro-1H-pyrido [1,2-b ] pyridazine-4, 6-dione

The specific reaction equation is as follows:

step A:3- (3- (benzyloxy) -4-oxo-4H-pyran-2-yl) -3-hydroxypropionic acid methyl ester (Compound A.1)

Methyl acetate (1.92g, 26mmol) was dissolved in 20mL tetrahydrofuran solution, LDA (0.5M,13mL) was added dropwise at-65 ℃ under nitrogen, and stirred for 1 hour. 3- (benzyloxy) -4-oxo-4H-pyran-2-carbaldehyde (2g, 8.66mmol) was added and stirred at-65 ℃ for 30 min. The reaction was quenched with saturated aqueous ammonium chloride solution, extracted three times with 90mL of ethyl acetate, and the organic phases were combined and dried over anhydrous sodium sulfate. Spin-drying and purification gave product A.1(1.54g, yield: 59%). LCMS ESI (+) m/z: 305.1(M +1).

And B: 3- (benzyloxy) -2- (1, 3-dihydroxypropyl) -4H-pyran-4-one (Compound A.2)

Compound A.1(1.5g, 4.93mmol) was dissolved in 15mL of tetrahydrofuran solution, and lithium borohydride (500mg, 13.34mmol) was added portionwise at 0 ℃ and stirred for 4 hours. The reaction was quenched with saturated aqueous ammonium chloride, extracted three times with 60mL ethyl acetate, the organic phases combined and dried over anhydrous sodium sulfate. Spin-drying and purification gave product A.2(1.1g, yield: 80%). LCMS ESI (+) m/z: 27.2(M +1).

And C: 3- (benzyloxy) -2- (3- ((tert-butyldimethylsilyl) oxy) -1-hydroxypropyl) -4H-pyran-4-one (Compound A.3)

Compound A.2(1.1g, 3.99mmol) was dissolved in 10mL of N, N-dimethylformamide, imidazole (596mg, 8.77mmol) was added, TBSCl (661mg, 4.38mmol) was added at 0 ℃ and stirred for 1 hour. The reaction was quenched with water, extracted three times with 60mL of ethyl acetate, and the organic phases were combined, spun-dried, and purified to give product A.3(600mg, yield: 38%). LCMSISI (+) m/z: 391.2(M +1).

Step D: 3- (benzyloxy) -2- (3- ((tert-butyldimethylsilyl) oxy) -1-hydroxypropyl) pyridin-4 (1H) -one (Compound A.4)

Compound A.3(1.8g,4.62mmol) was dissolved in 20mL of methanol solution, and ammonia (30mL) solution was added and stirred at room temperature for 36 hours. Reaction was monitored by LCMS to completion, extracted three times with 60mL ethyl acetate, and the organic phases were combined and dried over anhydrous sodium sulfate. Spin-drying and purification gave product A.4(560mg, yield: 31%). LCMS ESI (+) m/z: 390.2 (M +1).

Step E: 1-amino-3- (benzyloxy) -2- (3- ((tert-butyldimethylsilyl) oxy) -1-hydroxypropyl) pyridin-4 (1H) -one (Compound A.5)

Compound A.4(560mg, 1.44mmol) was dissolved in 8mL of N, N-dimethylformamide solution, followed by addition of 2, 4-dinitrophenylhydroxylamine (344mg, 1.73mmol) and potassium carbonate (993mg, 7.2 mmol). After stirring at room temperature for 2 hours, the reaction was quenched by addition of water (20mL), extracted three times with 50mL of ethyl acetate, and the organic phases were combined and dried over anhydrous sodium sulfate. Spin-drying and purification gave product A.5(480mg, yield: 82%). LCMS ESI (+) m/z: 405.2(M +1).

Step F: tert-butyl (3- (benzyloxy) -2- (3- ((tert-butyldimethylsilyl) oxy) -1-hydroxypropyl) -4-oxopyridin-1 (4H) -yl) carbamate (Compound A.6)

Compound A.5(200mg, 0.5mmol) was dissolved in 10mL of a tetrahydrofuran solution, and di-tert-butyl dicarbonate (119mg, 0.55mmol), triethylamine (100mg, 1mmol) and 4-dimethylaminopyridine (6mg, 0.05mmol) were added in this order, followed by stirring at room temperature for 30 minutes. After completion of the reaction by TLC, the reaction mixture was concentrated under reduced pressure to remove tetrahydrofuran, and the residue was diluted with water (20mL), extracted three times with 30mL of ethyl acetate, and the organic phases were combined and dried over anhydrous sodium sulfate. Filtration and purification by rotary drying gave product A.6(170mg, yield: 68%). LCMS ESI (+) m/z: 505.3(M +1).

Step G tert-butyl (3- (benzyloxy) -2- (1, 3-dihydroxypropyl) -4-oxopyridin-1 (4H) -yl) carbamate (Compound A.7)

Compound A.6(170mg, 0.34mmol) was dissolved in 5mL tetrahydrofuran solution and p-toluenesulfonic acid (128mg, 0.67mmol) was added at 0 ℃ and stirred for 2 h. The reaction mixture was diluted with water (10mL), extracted three times with 30mL ethyl acetate, and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and purified by spin-drying to give product A.7(140mg, yield: 95%). LCMS ESI (+) m/z: 391.2(M +1).

Step H: (3- (benzyloxy) -1- ((tert-butoxycarbonyl) amino) -4-oxo-1, 4-dihydropyridin-2-yl) -3-hydroxypropyl methanesulfonate (Compound A.8)

Compound A.7(140mg, 0.36mmol) was dissolved in 5mL of dichloromethane and DIPEA (56 mg, 0.43mmol) and MsCl (49mg, 0.43mmol) were added at 0 ℃ and stirred for 1 hour. The reaction was quenched with water, extracted three times with 30mL of dichloromethane, and the organic phases were combined and dried over anhydrous sodium sulfate. Filtration and purification by rotary drying gave product A.8(140mg, yield: 83%). LCMS ESI (+) m/z: 469.2(M +1).

Step I: 5- (benzyloxy) -4-hydroxy-6-oxo-2, 3,4, 6-tetrahydro-1H-pyrido [1,2-b ] pyridazine-1-carboxylic acid tert-butyl ester (Compound A.9)

Compound A.8(140mg, 0.30mmol) was dissolved in 5mL tetrahydrofuran solution, TBAF (1M,0.4mL) was added, and the mixture was stirred at room temperature overnight. After completion of the reaction, water (20mL) was added for dilution, extraction was carried out three times with 30mL of ethyl acetate, and the organic phases were combined and dried over anhydrous sodium sulfate. Filtration, spin-drying and purification gave product A.9(80mg, yield: 71%). LCMS ESI (+) m/z: 373.2(M +1).

Step J: 5- (benzyloxy) -4, 6-dioxo-2, 3, 6-trihydro-1H-pyrido [1,2-b ] pyridazine-1-carboxylic acid tert-butyl ester (Compound A.10)

Compound A.9(80m) was dissolved in 200mL of dichloromethane, cooled to 0 deg.C, 120mg of dess-martin oxidant was added slowly, warmed to room temperature and stirred for 1 hour. The reaction was quenched by adding sodium bicarbonate and aqueous sodium thiosulfate, extracted three times with 90mL of dichloromethane, the organic phases were combined, washed three times with saturated brine, and the organic phase was dried over anhydrous sodium sulfate. Filtration, spin-drying and purification gave product A.10(78mg, yield: 97%). LCMS ESI (+) M/z 371.2(M +1).

Step K: 5- (benzyloxy) -2, 3-dihydro-1H-pyrido [1,2-b ] pyridazine-4, 6-dione

Compound A.10(78mg,0.22mmol) was dissolved in 5mL of dichloromethane, and a solution of trifluoroacetic acid (2.5mL) was added thereto at 0 ℃ and stirred for 1 hour. The reaction was quenched by addition of saturated sodium bicarbonate solution at low temperature, extracted three times with 15mL of dichloromethane, the organic phases combined and dried over anhydrous sodium sulfate. Filtration, spin-drying, and purification by column chromatography gave intermediate A (45mg, yield: 77%). LCMS ESI (+) m/z: 271.2(M +1).

Intermediate B5- (benzyloxy) -3, 3-dimethyl-2, 3-dihydro-1H-pyrido [1,2-B ] pyridazine-4, 6-dione

The specific reaction equation is as follows:

step A3- (3- (benzyloxy) -4-oxo-4H-pyran-2-yl) -3-hydroxy-2, 2-dimethylpropionaldehyde (Compound B3.1)

3- (benzyloxy) -4-oxo-4H-pyran-2-carbaldehyde (10g,43mmol) was dissolved in 100mL of tetrahydrofuran, and pyrrolidine (0.35mL,17.5mmol), glacial acetic acid (3.73mL,97mmol) and isobutyraldehyde (4.3g,60mmol) were added at room temperature and stirred for 2 hours. The reaction solution was directly spin-dried and purified by column chromatography to give product B.1(9g, yield: 68.5%). LCMS ESI (+) M/z 303.1(M +1).

Step B5- (benzyloxy) -4-hydroxy-3, 3-dimethyl-3H-pyrido [1,2-B ] pyridazin-6 (4H) -one (Compound B.2)

Compound B.1(13g,43mmol) was dissolved in 200mL of N, N-dimethylformamide, hydrazine hydrochloride (30g,430mmol) was added, and the mixture was stirred at 80 ℃ for 2 hours. The reaction solution was poured into 1L of water, extracted three times with 500mL of ethyl acetate, the organic phases were combined, washed with saturated brine, and the organic phase was dried over anhydrous sodium sulfate, filtered, spin-dried, and purified by column chromatography to give product B.2(5g, yield: 38.8%). LCMS ESI (+) M/z 299.1(M +1).

Step C tert-butyl 5- (benzyloxy) -3, 3-dimethyl-6-oxo-4, 6-dihydro-3H-pyrido [1,2-b ] pyridazin-4-ylcarbonate (Compound B.3)

Compound B.2(12.5g,41mmol), triethylamine (6.35g,62.9mmol) and N, N-dimethyl-4-aminopyridine (512mg, 4.1mmol) were dissolved in 200mL of tetrahydrofuran, and a solution of di-tert-butyl dicarbonate (13.8g,63mmol) in 50mL of tetrahydrofuran was slowly added dropwise at zero degrees C, followed by stirring at room temperature for 1 hour. 500mL of water was added to the reaction mixture, and the mixture was extracted three times with 250mL of ethyl acetate, and the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, spin-dried, and purified to give product B.3(12.5g, yield: 74.9%, oily substance). LCMS ESI (+) M/z 399.1(M +1).

Step D tert-butyl 5- (benzyloxy) -3, 3-dimethyl-6-oxo-2, 3,4, 6-tetrahydro-1H-pyrido [1,2-b ] pyridazin-4-ylcarbonate (Compound B.4)

Compound B.3(12.5g,31.3mmol) was dissolved in 200mL of tetrahydrofuran solution, and sodium borohydride (2.2g,58.1mmol) was added in portions while cooling on ice, and stirred for 30 minutes. Quenched with saturated aqueous ammonium chloride solution, extracted three times with 250mL of ethyl acetate, the organic phases were combined, washed with saturated brine and dried over anhydrous sodium sulfate. Filtration, spin-drying and purification by column chromatography gave product B.4(12g, yield: 95.5%). LCMS ESI (+) M/z 401.1(M +1).

Step E (benzyloxy) -4- ((tert-butoxycarbonyl) oxy) -3, 3-dimethyl-6-oxo-2, 3,4, 6-tetrahydro-1H-pyrido (9H-fluoren-9-yl) o [1,2-b ] pyridazine-1-carboxylic acid tert-butyl ester (Compound B.5)

Compound B.4(5g,12.5mmol) was dissolved in 50mL of tetrahydrofuran solution, and fluorenylmethoxycarbonylcarbonyl chloride (3.9g, 15.1mmol) and a saturated aqueous solution of sodium hydrogencarbonate (10mL) were added under ice-bath, and the mixture was warmed to room temperature and stirred for 0.5 hour. The reaction was quenched with 150mL of water, extracted three times with 100mL of ethyl acetate, the organic phases were combined, washed three times with saturated brine, and the organic phase was dried over anhydrous sodium sulfate, filtered, spun-dried, and purified to give product B.5(7.9g, yield: 67%). LCMS ESI (+) M/z 622.2(M +1).

Step F (9H-fluoren-9-yl) methyl 5- (benzyloxy) -4-hydroxy-3, 3-dimethyl-6-oxo-2, 3,4, 6-tetrahydro-1H-pyrido [1,2-b ] pyridazine-1-carboxylic acid tert-butyl ester (Compound B.6)

Compound B.5(7.9g,12.7mmol) was dissolved in 40mL of dichloromethane and trifluoroacetic acid (20mL) was added dropwise under ice-bath, slowly warmed to room temperature and stirred for 30 min. The reaction was directly spin-dried to give product B.6(6g, yield: 98%). LCMS ESI (+) M/z 523.2(M +1).

Step G (9H-fluoren-9-yl) methyl 5- (benzyloxy) -3, 3-dimethyl-4, 6-dioxo-2, 3,4, 6-tetrahydro-1H-pyrido [1,2-b ] pyridine-1-carboxylic acid (compound B.7)

Compound B.6(6g,11.5mmol) was dissolved in 200mL of dichloromethane, cooled to 0 deg.C, slowly added 9.8g of dess-martin oxidant, warmed to room temperature and stirred for 1 hour. The reaction was quenched by adding sodium bicarbonate and aqueous sodium thiosulfate, extracted three times with 200mL of dichloromethane, the organic phases were combined, washed three times with saturated brine, and the organic phase was dried over anhydrous sodium sulfate. Filtration, spin-drying and purification gave B.7(5.5g, yield: 98%). LCMS ESI (+) M/z:521.2(M +1).

Step H5- (benzyloxy) -3, 3-dimethyl-2, 3-dihydro-1H-pyrido [1,2-B ] pyridazine-4, 6-dione (intermediate B)

Compound B.7(5.5g,10.6mmol) was dissolved in 55mL of dichloromethane solution, 5.5mL of piperidine was added, the reaction was stirred at room temperature for 1 hour, water was added to quench the reaction, three times extraction was performed with 200mL of dichloromethane, the organic phases were combined, washed three times with saturated brine, and the organic phase was dried over anhydrous sodium sulfate. Filtration, spin-drying and purification by column chromatography gave intermediate B (2.8g, yield: 94%). LCMS ESI (+) M/z 299.1(M +1).¹H NMR(400MHz,DMSO-d6)δ7.74(d,J＝ 7.6Hz,1H),7.49–7.41(m,2H),7.35(dd,J＝8.5,3.6Hz,3H),7.01(t,J＝7.1 Hz,1H),6.23(d,J＝7.6Hz,1H),5.16(s,2H),3.20(d,J＝7.1Hz,2H),1.11(s, 6H).

Intermediate C: 5'- (benzyloxy) -1', 2 '-dihydrospiro [ cyclopropane-1, 3' -pyrido [1,2-b ] pyridazine ] -4',6' -dione

The specific reaction equation is as follows:

step A1- ((3- (benzyloxy) -4-oxo-4H-pyran-2-yl) (hydroxy) methyl) cyclopropanecarboxaldehyde (compound C.1)

3- (benzyloxy) -4-oxo-4H-pyran-2-carbaldehyde (8g,30.4mmol) was dissolved in 400mL of tetrahydrofuran, and pyrrolidine (0.2mL, 10mmol), glacial acetic acid (1.73mL, 45mmol) and cyclopropylformaldehyde (3g, 36.5mmol) were added at room temperature and stirred for 2 hours. The reaction solution was directly spin-dried and purified by column chromatography to obtain product C.1(1.1g, yield: 12%). LCMS ESI (+) M/z:301.1(M +1).

And B: 4', 5' -Dihydroxyspiro [ cyclopropane-1, 3' -pyrido [1,2-b ] pyridazine ] -6' (4' H) -ketoxazine ] -6' (4' H) -one (Compound C.2)

Compound C.1(1.1g, 3.67mmol) was dissolved in 20mL of N, N-dimethylformamide, hydrazine hydrochloride (2.5g,36.7mmol) was added, and the mixture was stirred at 80 ℃ for 2 hours. The reaction solution was poured into 100mL of water, extracted three times with 60mL of ethyl acetate, the organic phases were combined, washed with saturated brine, and the organic phase was dried over anhydrous sodium sulfate. Filtration, spin-drying and purification by column chromatography gave product C.2(670mg, yield: 62%). LCMS ESI (+) M/z:297.1(M +1).

And C: 5'- (benzyloxy) -6' -oxo-4 ',6' -dihydrospiro [ cyclopropane-1, 3 '-pyrido [1,2-b ] pyridazin ] -4' -yl ester (Compound C.3)

Compound C.2(670mg, 2.26mmol), triethylamine (461mg, 4.52mmol) and N, N-dimethyl-4-aminopyridine (83mg, 0.68mmol) were dissolved in 10mL of tetrahydrofuran, and a solution of acetic anhydride (462mg, 4.52mmol) in 3mL of tetrahydrofuran was slowly dropped at 0 ℃ and then stirred at room temperature for 1 hour. 10mL of water was added to the reaction mixture, and the mixture was extracted three times with 30mL of ethyl acetate, and the organic phases were combined, washed with saturated brine, and dried over anhydrous sodium sulfate. Filtration, spin-drying and column purification gave the product C.3 as an oil (800mg, yield: 99%). LCMS ESI (+) M/z 339.1(M +1).

Step D: 5'- (benzyloxy) -6' -oxo-1 ', 2', 4',6' -tetrahydrospiro [ cyclopropane-1, 3 '-pyrido [1,2-b ] pyridin ] -4' -yl ester (Compound C.4)

Compound C.3(800mg, 3.2mmol) was dissolved in 20mL of tetrahydrofuran solution, and sodium borohydride (171mg, 5.3mmol) was added in portions while cooling on ice, and stirred for 30 minutes. Quenched with saturated ammonium chloride solution, extracted three times with 30mL of ethyl acetate, the organic phases were combined, washed with saturated brine and dried over anhydrous sodium sulfate. Filtered, spun-dried and purified to give product C.4(725mg, yield: 90%). LCMS ESI (+) M/z 341.1(M +1).

Step E4 ' -acetoxy-5 ' - (benzyloxy) -6' -oxo-4 ',6' -dihydrospiro [ cyclopropane-1, 3' -pyrido [1,2-b ] pyridazine ] -1'1H) -carboxylic acid tert-butyl ester (Compound C.5)

Compound C.4(900mg, 2.64mmol), triethylamine (342mg, 5.3mmol) and N, N-dimethyl-4-aminopyridine (28mg, 0.23mmol) were dissolved in 10mL of tetrahydrofuran, and a solution of di-tert-butyl dicarbonate (641mg, 5.3mmol) in 3mL of tetrahydrofuran was slowly dropped at 0 ℃ and then stirred at room temperature for 1 hour. The reaction mixture was diluted with 20mL of water, extracted three times with 30mL of ethyl acetate, the organic phases were combined, washed with saturated brine, and the organic phase was dried over anhydrous sodium sulfate. Filtration, spin-drying and column purification gave product C.5 as an oil (1.1g, yield: 95%). LCMS ESI (+) M/z:441.1(M +1).

Step F: (benzyloxy) -4 '-hydroxy-6' -oxo-4 ',6' -dihydrospiro [ cyclopropane-1, 3 '-pyrido [1,2-b ] pyridazine ] -1' (21H) -carboxylic acid tert-butyl ester (Compound C.6)

Compound C.5(1.1g, 2.50mmol) was dissolved in 20mL of methanol solution, potassium carbonate (312mg,2.26mmol) was added, and the mixture was stirred at room temperature for 1 hour. After completion of the reaction, the reaction mixture was concentrated under reduced pressure, and the residue was diluted with water (30mmL), extracted twice with 40mL of ethyl acetate, and the organic phase was dried over anhydrous sodium sulfate. Filtration, spin-drying and purification gave product C.6(550mg, yield: 55%). LCMS ESI (+) M/z 399.1(M +1).

Step G: (benzyloxy) -4',6' -dioxo-4 ',6' -dihydrospiro [ cyclopropane-1, 3' -pyrido [1,2-b ] pyridazine ] -1' (2' H) -carboxylic acid tert-butyl ester (Compound C.7)

Compound C.6(550mg, 1.38mmol) was dissolved in 10mL of dichloromethane, cooled to 0 deg.C, slowly added 1.4g of dess-martin oxidant, gradually warmed to room temperature and stirred for 1 hour. The reaction was quenched by adding sodium bicarbonate and aqueous sodium thiosulfate, extracted three times with 20mL of dichloromethane, the organic phases were combined, washed three times with saturated brine, and the organic phase was dried over anhydrous sodium sulfate. Filtration, spin-drying and purification gave product C.7(500mg, yield: 91%). LCMS ESI (+) M/z 397.1(M +1).

Step H: 5'- (benzyloxy) -1', 2 '-dihydrospiro [ cyclopropane-1, 3' -pyrido [1,2-b ] pyridazine ] -4',6' -dione (intermediate C)

Compound C.7(500mg, 1.26mmol) was dissolved in 10mL of dichloromethane, 3mL of trifluoroacetic acid was slowly added dropwise in an ice bath, and after completion of addition, the mixture was warmed to room temperature and stirred for 30 minutes. The reaction solution was directly spin-dried to obtain intermediate C (100mg, yield: 27.1%). LCMS ESI (+) M/z:297.1(M +1).

The procedure for the synthesis of intermediate C was used to synthesize the intermediates listed in the table below

TABLE 1

Intermediate series 2

Intermediate I: 7, 8-difluoro-6, 11-dihydrodibenzo ([ b, e ] thiaheptacyclo-1, 2,3,4-d 4-11-ol

The specific synthesis steps are as follows:

step A: thiophenol-d 5 (Compound I.1)

Magnesium turnings (4.4g, 0.18mol), 1, 2-dibromoethane (1mL) were added to 100mL tetrahydrofuran solution, bromobenzene-d 5(2g,12.3mmol) was added dropwise under nitrogen, after the reaction was initiated, bromobenzene d5(18g, 0.11mol) in 100mL tetrahydrofuran was slowly added dropwise to the reaction solution, and the mixture was refluxed at 70 ℃ for one hour. Then, the temperature is reduced to room temperature, 5.5g of sulfur powder is added in portions, and the reaction is stirred for one hour. The reaction solution was filtered, the filtrate was quenched with concentrated hydrochloric acid, the aqueous phase was extracted twice with 100mL of ethyl acetate, the organic phases were combined, the organic phase was washed with 100mL of saturated brine, and dried over anhydrous sodium sulfate. Filtration and spin-drying gave the crude product I.1(14g,0.12mol) as a yellow oil.

And B: methyl 3, 4-difluoro-2- ((phenylthio-d 5) methyl) benzoate (Compound I.2)

Methyl 2- (bromomethyl) -3, 4-difluorobenzoate (30.6g,0.12mol) was dissolved in N, N-dimethylformamide (150mL), and compound I.1(14g,0.12mol) and potassium carbonate (21.8g, 0.16mol) were added under ice-bath and stirred at 0 ℃ for 1 hour. After completion of the reaction, the reaction mixture was poured into 1L of water, extracted three times with 200mL of ethyl acetate, and the organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. Filtration, spin-drying and purification by column chromatography gave product i.2 as a colourless oil (20.7g, 59.8%). LCMS ESI (+) M/z:300.1(M +1).

And C: 3, 4-difluoro-2- ((phenylthio-d 5) methyl) benzoic acid (Compound I.3)

Compound I.2(20.7g, 0.069mol) was dissolved in 100mL of tetrahydrofuran and 15mL of methanol, and aqueous sodium hydroxide (6N, 100mL) was added thereto at room temperature, followed by stirring at room temperature overnight. The reaction mixture was neutralized with concentrated hydrochloric acid to pH 5, extracted twice with 200mL of ethyl acetate, and the organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. Filtration and spin-drying gave compound I.3(19.2g, yield: 99.3%) as a white solid. LCMS ESI (+) M/z 286.1(M +1).

Step D: 7, 8-Difluorodibenzo [ b, e ] thiaheptacyclo-11 (6H) one-1, 2,3,4-d4 (Compound I.4)

Compound I.3(19.1g,66.9mmol) was dissolved in 40g of polyphosphoric acid and 150mL of sulfolane, warmed to 100 ℃ and the reaction stirred for 2 hours. Water (400mL) was added to the reaction solution, which was extracted with ethyl acetate (200mLX 3). The organic phase was washed with saturated sodium bicarbonate solution (200mL) and dried over anhydrous sodium sulfate. Filtration and spin-drying gave compound I.4 as a yellow solid (10.8g, yield: 60.7%). LCMS ESI (+) M/z 267.1(M +1).¹HNMR(400MHz,DMSO-d6)δ 7.51-7.38(m,2H),4.34(d,J＝1.0Hz,2H).

Step E: 7, 8-difluoro-6, 11-dihydrodibenzo ([ b, e ] thiaheptacyclo-1, 2,3,4-d 4-11-ol (intermediate I)

Compound 1.4(100mg,0.38mmol) was dissolved in a tetrahydrofuran/methanol (5mL/1mL) mixed solution under ice-bath conditions, followed by addition of sodium borohydride (21mg, 0.56mmol) and stirring at zero degrees for an additional 1 hour. After the reaction was complete, 2.5mL of saturated ammonium chloride solution was added and quenched. Extract 2 times with 15mL ethyl acetate and combine the organic phases. The organic phase was washed with 20mL of saturated brine. Concentration by drying gave compound intermediate I as a white solid (102mg, yield: 99%). LCMS ESI (+) M/z 269.1(M +1).

The procedure for the synthesis of intermediate I was used for the synthesis of the intermediates listed in the table below

TABLE 2

Example 1

1'- (1,2,3, 4-tetradeutero-7, 8-difluoro-6, 11-dihydrodibenzo [ b, e ] thiepin-11-yl) -5' -hydroxy-1 ", 2 '-dihydrospiro [ cyclobutane-1, 3' -pyrido [1,2, 2-b ] pyridazine ] -4',6' -dione

The specific reaction equation is as follows:

step A: 5' - (benzyloxy) -1' - (7, 8-difluoro-6, 11-dihydrodibenzo [ b, e ] thiepin-11-yl-1, 2,3,4-D4) -1', 2' -dihydrospiro [ cyclobutane-1, 3' -pyrido [1,2-b ] pyridazine ] -4',6' -dione (Compound 1.1)

Intermediate D (130mg,0.42mmol) was dissolved in N, N-dimethylformamide (0.5mL), and then intermediate J (102.7mg,1.52mmol) and a solution of T3P/DMF (50%) (5mL) were added to the reaction solution, and the reaction was stirred at 100 ℃ for 1 hour. The reaction was quenched with saturated sodium bicarbonate solution (5mL), the aqueous phase was extracted 2 times with ethyl acetate (15mL), and the organic phases were combined. The organic phase was dried over anhydrous sodium sulfate. Filtration, spin-drying and purification gave compound 1.1(100mg, yield: 41.8%) as a yellow solid. LCMS ESI (+) M/z 561.2(M +1).

Step B1 '- (7, 8-difluoro-6, 11-dihydrodibenzo [ B, e ] thiepin-11-yl-1, 2,3,4-d4) -5' -hydroxy-1 ', 2' -dihydrospiro [ cyclobutane-1, 3 '-pyrido [1,2-B ] pyridazine ] -4, 6' -dione (Compound 1)

Compound 1.1(100mg,0.18mmol) was dissolved in 5mL of N, N-dimethylformamide, lithium chloride (100mg,2.36mmol) was added, and the mixture was heated to 100 ℃ and reacted for 1 h. To the reaction mixture were added 10mL of water and 10mL of ethyl acetate, and the mixture was stirred, allowed to stand for separation, the aqueous layer was extracted with ethyl acetate (10 mL. times.3), and the organic phases were combined. The extract was washed with saturated brine (50mL) and dried over anhydrous sodium sulfate. Filtration, spin-drying, and purification of the crude product by reverse phase preparative lyophilization afforded Compound 1 as a yellow solid (22mg, yield: 26.3%). LCMS ESI (+) M/z:471.1(M +1).

Example 2

Compound example 2 was obtained from intermediate a and intermediate I in a two-step reaction using the same reaction conditions of example 1.

1- (7, 8-difluoro-1, 2,3, 4-tetradeuterium-6, 11-dihydrodibenzo [ b, e ] thiepin-11-yl) -5-hydroxy-2, 3-dihydro-1H-pyrido [1,2-b ] pyridazine-4, 6-dione

LCMS ESI(+)m/z:431.1(M+1).

Compounds example 3 and example 4 were synthesized from intermediate B, intermediate F and intermediate I by a two-step reaction using the same reaction conditions as in example 1:

example 3

1- (7, 8-difluoro-1, 2,3, 4-tetradeuterium-6, 11-dihydrodibenzo [ b, e ] thiepin-11-yl-1, 2,3,4-d4) -5-hydroxy-3, 3-dimethyl-2, 3-dihydro-1H-pyrido [1,2b ] pyridazine-4, 6-dione

LCMS ESI(+)m/z:459.1(M+1).

Example 4

1'- (7, 8-difluoro-6, 11-dihydrodibenzo [ b, e ] thiepin-11-yl-1, 2,3,4-d4) -5' -hydroxy-1 ", 2 '-dihydrospiro [ cyclopentane-1, 3' -dihydropyrido [1,2, 2-b ] pyridazine ] -4',6' -dione

LCMS ESI(+)m/z:485.2(M+1).

Example 5

1- (-6, 11-dihydrodibenzo [ b, e ] thiepin-d-11-yl) -5-hydroxy-3, 3-dimethyl-2, 3-dihydro-1H-pyrido [1,2-b ] pyridazine-4, 6-dione

The specific reaction equation is as follows:

step A: 6, 11-Dihydrodibenzo [ b, e ] thiepin-d-11-ol (Compound 5.1)

The compound dibenzo [ b, e ] thiaheptacyclo-11 (6H) -one (100mg, 0.44mmol) was weighed out, dissolved in 5mL tetrahydrofuran and 1mL methanol, and sodium borodeuteride (28mg, 0.663mmol) was added in portions, and stirred at 20 ℃ for 1 hour. To the reaction solution was added a saturated ammonium chloride solution (10mL), extracted 2 times with ethyl acetate (30mL), and the combined organic phases were washed 2 times with saturated brine (15mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give the product 5.1(100mg, yellow solid, yield: 99%).

And B:5- (benzyloxy) -1- (6, 11-dihydrodibenzo [ b, e ] thio-d-11-yl) -3, 3-dimethyl-2, 3-dihydro-1H-pyrido [1,2-b ] pyridazine-4, 6-dione (Compound 5.2)

Compound 5.1(100mg, 0.436mmol) and compound B (100mg, 0.335mmol) were dissolved in 4mL 1-propylphosphoric anhydride/N, N-dimethylformamide (50%). The reaction solution was stirred at 100 ℃ under nitrogen for 1 hour. Water (20mL) was added to the reaction mixture, which was extracted 2 times with ethyl acetate (30mL), and the combined organic phases were washed 3 times with saturated brine (15mL) and dried over anhydrous sodium sulfate. Filtration and concentration under reduced pressure, the residue was purified by column chromatography (petroleum ether/ethyl acetate 0/1) to give 5.2(122mg, white solid, yield: 71%). LCMS ESI (+) M/z 510.2(M +1).

And C: 1- (-6, 11-dihydrodibenzo [ b, e ] thiepin-d-11-yl) -5-hydroxy-3, 3-dimethyl-2, 3-dihydro-1H-pyrido [1,2-b ] pyridazine-4, 6-dione (Compound 5)

Compound 5.2(50mg, 0.098mmol) was dissolved in 3mL of N, N-dimethylformamide, and lithium chloride (83mg, 1.96mmol) was added. The reaction solution was stirred at 100 ℃ under nitrogen for 4 hours. The reaction solution was concentrated under reduced pressure, and the residue was purified by reverse phase preparation to give the product (36mg, pale yellow solid, yield: 88%). LCMS ESI (+) M/z:420.1(M +1).

¹H NMR(400MHz,DMSO-d6)δ7.47-7.34(m,4H),7.30-7.22(m,1H),7.13-7.02 (m,2H),6.87-6.78(m,2H),5.75(d,J＝7.6Hz,1H),5.66(d,J＝13.2Hz,1H), 3.87(d,J＝13.2Hz,1H),3.64(d,J＝14.8Hz,1H),3.24(d,J＝14.8Hz,1H), 1.38(s,3H),1.07(s,3H)

Example 6

1- (7, 8-difluoro-6, 11-dihydrodibenzo [ b, e ] thiepin-d 2-11-yl) -5-hydroxy-3, 3-dimethyl-2, 3-dihydro-1H-pyrido [1,2b ] pyridazine-4, 6-dione

The specific reaction equation is as follows:

step A: 7, 8-Difluorodibenzo [ b, e ] thiaheptacyclo-d 2-11(6H) -one (Compound 6.1)

The compound 7, 8-difluorodibenzo [ b, e ] thiaheptacyclo-11 (6H) -one (100mg, 0.381mmol) was dissolved in 4mL of deuteromethanol and 1mL of 1, 2-dichloroethane, and sodium methoxide (83mg, 1.53mmol) was added thereto, followed by stirring at 50 ℃ under nitrogen for 12 hours. Water (10mL) was added to the reaction mixture, and the mixture was extracted 2 times with ethyl acetate (30 mL). The combined organic phases were washed 2 times with saturated brine (15mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by column chromatography (petroleum ether/ethyl acetate 20/1) to give the product (60mg, white solid, yield: 60%).

And B: 7, 8-difluoro-6, 11-dihydrodibenzo [ b, e ] thiepin-d 2-11-ol (Compound 6.2)

Compound 6.1(60mg, 0.227mmol) was weighed out and dissolved in 4mL of tetrahydrofuran and 1mL of methanol, and sodium borohydride (13mg, 0.340mmol) was added in portions and stirred at 0 ℃ for 1 hour. To the reaction solution was added a saturated ammonium chloride solution (10mL), extracted 2 times with ethyl acetate (30mL), and the combined organic phases were washed 2 times with saturated brine (15mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give the product (55mg, white solid, yield: 92%).

And C:5- (benzyloxy) -1- (7, 8-difluoro-6, 11-dihydrodibenzo [ b, e ] thio-d 2-11-yl) -3, 3-dimethyl-2, 3-dihydro-1H-pyrido [1,22-b ] pyridazine-4, 6-dione (Compound 6.3)

Compound 6.2(55mg, 0.205mmol) and compound B (51mg, 0.171mmol) were dissolved in 3ml of 1-propylphosphoric anhydride/N, N-dimethylformamide 50%. The reaction solution was stirred at 100 ℃ under nitrogen for 1 hour. To the reaction solution was added water (20ml), which was extracted 2 times with ethyl acetate (30ml), and the combined organic phases were washed 3 times with saturated brine (15ml), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by column chromatography (petroleum ether/ethyl acetate: 0/1) to give the product (40mg, white solid, yield: 43%). LCMS ESI (+) M/z 547.2(M +1).

Step D: 1- (7, 8-difluoro-6, 11-dihydrodibenzo [ b, e ] thiepin-d 2-11-yl) -5-hydroxy-3, 3-dimethyl-2, 3-dihydro-1H-pyrido [1,2b ] pyridazine-4, 6-dione (Compound 6)

Compound 6.3(40mg, 0.073mmol) was dissolved in 3ml of N, N-dimethylformamide, and lithium chloride (62mg, 1.46mmol) was added. The reaction solution was stirred at 100 ℃ under nitrogen for 3 hours. The reaction solution was concentrated under reduced pressure, and the residue was purified by reverse phase preparation to give the product (20mg, pale yellow solid, yield: 60%). LCMS ESI (+) M/z:457.1(M +1).

¹H NMR(400MHz,DMSO-d6)δ7.49-7.27(m,3H),7.18-7.00(m,2H),6.92-6.77 (m,2H),5.73(d,J＝7.6Hz,1H),5.29(s,1H),3.63(d,J＝15.2Hz,1H),3.31 (d,J＝15.2Hz,1H),1.38(s,3H),1.07(s,3H)

Example 7

1- (7, 8-difluoro-6, 11-dihydrodibenzo [ b, e ] thiepin-d 3-11-yl) -5-hydroxy-3, 3-dimethyl-2, 3-dihydro-1H-pyrido [1,2b ] pyridazine-4, 6-dione

The specific reaction equation is as follows:

step A: 7, 8-difluoro-6, 11-dihydrodibenzo [ b, e ] thiepin-d 3-11-ol (Compound 7.1)

Compound 6.1(134mg, 0.507mmol) was weighed out, dissolved in 4mL of tetrahydrofuran and 1mL of methanol, added portionwise with sodium borodeuteride (28mg, 0.66mmol) and stirred at 20 ℃ for 1 hour. To the reaction solution was added a saturated ammonium chloride solution (10mL), extracted 2 times with ethyl acetate (30mL), and the combined organic phases were washed 2 times with saturated brine (15mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give the product (130mg, white solid, yield: 96%).

And B:5- (benzyloxy) -1- (7, 8-difluoro-6, 11-dihydrodibenzo [ b, e ] thio-d 3-11-yl) -3, 3-dimethyl-2, 3-dihydro-1H-pyrido [1,22-b ] pyridazine-4, 6-dione (Compound 7.2)

Compound 7.1(130mg, 0.48mmol) and compound 3.3(120mg, 0.40mmol) were dissolved in 4ml of 1-propylphosphoric anhydride/N, N-dimethylformamide 50%. The reaction solution was stirred at 100 ℃ under nitrogen for 1 hour. Water (20ml) was added to the reaction solution, extracted 2 times with ethyl acetate (30ml), the combined organic phases were washed 3 times with saturated brine (15ml), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by column chromatography (petroleum ether/ethyl acetate: 0/1) to give the product (110mg, white solid, yield: 50%). LCMS ESI (+) M/z 548.2(M +1).

And C: 1- (7, 8-difluoro-6, 11-dihydrodibenzo [ b, e ] thiepin-d 3-11-yl) -5-hydroxy-3, 3-dimethyl-2, 3-dihydro-1H-pyrido [1,2b ] pyridazine-4, 6-dione (Compound 7)

Compound 7.2(110mg, 0.200mmol) was dissolved in 3ml of N, N-dimethylformamide, and lithium chloride (170mg, 4.02mmol) was added. The reaction solution was stirred at 100 ℃ under nitrogen for 2 hours. The reaction solution was concentrated under reduced pressure, and the residue was purified by reverse phase preparation to give the product (47mg, pale yellow solid, yield: 51.1%). LCMS ESI (+) M/z 458.1(M +1).

¹H NMR(400MHz,DMSO-d6)δ7.46-7.29(m,3H),7.20-7.00(m,2H),6.92-6.80 (m,2H),5.75(d,J＝7.6Hz,1H),3.64(d,J＝14.8Hz,1H),3.32(d,J＝14.8Hz,1 H),1.39(s,3H),1.08(s,3H)

Example 8

1' - (7, 8-difluoro-6, 11-dihydrodibenzo [ b, e ] thiepin-D2-11-yl-1, 2,3,4-D4) -5' -hydroxy-1 ', 2' -dihydrospiro [ cyclobutane-1, 3' -pyrido [1,2, 2-b ] pyridazine ] -4',6' -dione was synthesized in four steps from intermediate I and intermediate D using the same conditions as compound 6 to give example 8 LCMS ESI (+) M/z:473.2(M +1).

Example 9

(S) -1'- (1,2,3, 4-tetradeutero-7, 8-difluoro-6, 11-dihydrodibenzo [ b, e ] thiepin-11-yl) -5' -hydroxy-1 ", 2 '-dihydrospiro [ cyclobutane-1, 3' -pyrido [1,2, 2-b ] pyridazine ] -4',6' -dione

The specific reaction is as follows:

step A: compound 1.2(1.10g,2.02mmol) was subjected to chiral separation to give compound 9.1(500mg, 45%).

Chiral separation conditions:

chiral column ChiralCel OD-H (Daicel chemical Industries, Ltd,250 x 30mm i.d.,5 um); mobile phase A is supercritical CO2, mobile phase B is isopropanol (A: B: 60: 40); flow rate: 50 ml/min; the column temperature is 38 ℃; detection wavelength of 220nm

Step B difluoro-6, 11-dihydrodibenzo [ B, e ] thien-11-yl) -5-hydroxy-3, 3-dimethyl-2, 3-dihydro-1H-pyrido [1,2-B ] pyridazine-4, 6-dione (Compound 9)

Compound 9.1(50mg,0.089mmol) was dissolved in DMF (0.5mL), lithium chloride (16mg,0.37mmol) was added, and the mixture was stirred at 100 ℃ for 5 hours. The solution was cooled to room temperature and purified directly by reverse phase preparative lyophilization to give example 9(18mg, 42.9%). LCMS ESI (+) M/z:471.1(M +1).

SFC separation and debenzylation were performed on the samples 3 and 4 under the same separation and synthesis conditions as in example 9 to obtain examples 10 and 11

Example 10

(S) -1- (7, 8-difluoro-6, 11-dihydrodibenzo [ b, e ] thiepin-11-yl-1, 2,3,4-d4) -5-hydroxy-3, 3-dimethyl-2, 3-dihydro-1H-pyrido [1,2b ] pyridazine-4, 6-dione

LCMS ESI(+)m/z:459.1(M+1).

Example 11

(S) -1'- (7, 8-difluoro-6, 11-dihydrodibenzo [ b, e ] thiepin-11-yl-1, 2,3,4-d4) -5' -hydroxy-1 ", 2 '-dihydrospiro [ cyclopentane-1, 3' -dihydropyrido [1,2, 2-b ] pyridazine ] -4',6' -dione

LCMS ESI(+)m/z:485.2(M+1)。

Example 12

(S) - ((1' - (7, 8-difluoro-6, 11-dihydro-1, 2,3, 4-tetradeuterodibenzo [ b, e ] thiepin-11-yl) -4',6' -dioxo-1 ', 2', 4',6' -tetrahydrospiro [ cyclobutane-1, 3' -pyrido [1,2-b ] pyridazine ] -5' -yl) oxy) methyl methylcarbonate methyl ester

The specific synthetic route is shown in the following equation:

step A: chloromethyl methyl carbonate (Compound 12.1)

The weighed chloromethyl chloroformate (2g, 15.5mmol) was dissolved in dichloromethane (15mL), and then the reaction flask was placed under an ice bath, followed by addition of methanol (992mg, 31mmol) and pyridine (1.84g, 23.25mmol) to the reaction solution, which was then stirred at room temperature for 3 hours. Completion of the reaction was confirmed by TLC (no fluorescence, with potassium permanganate developer). The reaction mixture was diluted with dichloromethane (10mL), washed with a saturated sodium bicarbonate solution, a 1mol/L hydrochloric acid solution, a saturated sodium bicarbonate solution and a saturated brine, respectively, and the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated. Finally, crude compound 12.1(1.1g, yield: 56.7%, pale yellow oil) was obtained.

And B: iodomethyl methyl carbonate (Compound 12.2)

Compound 12.1(1.1g, 8.8mmol) was dissolved in acetone (15mL), then sodium iodide (3.96g, 26.4mmol) was added to the reaction solution, and the reaction flask was stirred in an oil bath at 55 deg.C for 2 hours. After the reaction was complete, the solvent was spun off with a water pump, the residue was dissolved in water (20mL), the aqueous phase was extracted with n-hexane, the combined organic phases were washed with saturated brine, dried and spun off. Finally, 12.2(890mg, yield: 46.8%, brown-yellow liquid) was obtained as a crude product.

And C: (S) - ((1' - (7, 8-difluoro-1, 2,3, 4-tetradeuterium-6, 11-dihydrodibenzo [ b, e ] thiepin-11-yl) -4',6' -dioxo-1 ', 2', 4',6' -tetrahydrospiro [ cyclobutane-1, 3' -pyrido [1,2-b ] pyridazine ] -5' -yl) oxy) methyl methylcarbonate (Compound 12)

The weighed compound, example 9(60mg, 0.128mmol), was dissolved in acetone (6mL), and cesium carbonate (85.4mg, 0.262mmol) and compound 12.2(84.89mg, 0.393mmol) were added to the reaction flask and stirred at room temperature overnight. Completion of the reaction was confirmed by LCMS. The reaction solvent was spun off with a water pump, the residue was dissolved in ethyl acetate (15mL), the organic phase was washed with water (10mL) and saturated brine (10mL), respectively, dried over anhydrous sodium sulfate, filtered, concentrated, and the crude product was dissolved in methanol (3mL) and then preparative purified by a reverse preparative instrument to give the compound example 12(24mg, yield: 33.5%, white solid). LCMS ESI (+) M/z 559.2(M +1).

The synthesis of example 12 was used to synthesize the following experimental examples.

TABLE 3

Experimental example 34

1'- (1,2,3, 4-tetradeutero-8-fluoro-6, 11-dihydrodibenzo [ b, e ] thiepin-11-yl) -5' -hydroxy-1 ", 2 '-dihydrospiro [ cyclopropane-1, 3' -pyrido [1,2, 2-b ] pyridazine ] -4',6' -dione

Synthesis of experiment 34 Using intermediate C and intermediate K, Using the procedure of Synthesis example 2

LCMS ESI(+)m/z:439.1(M+1).

Experimental example 35

((1- (8-fluoro-6, 11-dihydro-1, 2,3, 4-tetradeuterodibenzo [ b, e ] thiepin-11-yl) -4',6' -dioxo-1 ', 2', 4',6' -tetrahydrospiro [ cyclopropane-1, 3 '-pyrido [1,2-b ] pyridazine ] -5' -yl) oxy) methyl methylcarbonate

Experimental example 34 was converted to Experimental example 35 by the method of Synthesis of Experimental example 12

LCMS ESI(+)m/z:527.2(M+1).

Example 36

1'- (1,2,3,4,7,8,9, 10-octadeutero-6, 11-dihydrodibenzo [ b, e ] thiepin-11-yl) -5' -hydroxy-1 ", 2 '-dihydrospiro [ cyclopentane-1, 3' -pyrido [1,2, 2-b ] pyridazine ] -4',6' -dione

Synthesis of Experimental example 36 Using intermediate D and intermediate K, the procedure used to synthesize Experimental example 1 was used

LCMS ESI(+)m/z:453.2(M+1).

Example 37.

(1' - (1,2,3,4,7,8,9, 10-octadeutero-6, 11-dihydrodibenzo [ b, e ] thiepin-11-yl) -4',6' -dioxo-1 ', 2', 4',6' -tetrahydrospiro [ cyclopentane-1, 3' -pyrido [1,2-b ] pyridazine ] -5' -yl) oxy) methyl carbonate

Experimental example 36 was converted to Experimental example 37 using the method of synthetic Experimental example 12.

LCMS ESI(+)m/z:555.2(M+1).

Experimental example 38: inhibiting viral proliferative Activity (EC)₅₀) And Cytotoxicity (CC) of the Compound₅₀)

1) Cell treatment: MDCK cells were seeded in 384-well plates at a certain density, and then the cells were cultured overnight in a 5% CO2 incubator at 37 ℃.

2) Compound treatment: the test compound will be diluted in DMSO at a multiple ratio and added to the cell culture plate.

3) Virus inoculation: diluted influenza A/Weiss/43(H1N1) virus solution was then added to the antiviral activity assay wells, and no virus was added to the cytotoxicity assay wells. The final concentration of DMSO was 0.5%. Cells were incubated at 37 ℃ in a 5% CO2 incubator for 5 days until the cellular morbidity in virus control wells (no compound) reached 80-95%.

4) And (3) detecting the activity of the cells: adding a cell activity detection reagent CCK-8 into each hole, culturing cells in a 5% CO2 incubator at 37 ℃ for 3-4 hours, detecting a signal value by using a spectrophotometer, and calculating the activity and cytotoxicity of the compound against influenza virus according to original data. Compound dose response curves and their EC50 and CC50 values will be obtained after analysis by GraphPad Prism software. .

The activity of the partial compounds tested in this way to inhibit virus proliferation (EC50) and the cytotoxicity (CC50) of the compounds are listed in the following table:

experimental example 39: experimental example 11 Biggee pharmacokinetic experiment of the product

Preparation of intravenous administration solution of the product of Experimental example 11: about 40mg of each of Experimental example 11 was accurately weighed, dissolved in 10% by volume of N, N-Dimethylacetamide (DMA), added with 10% by volume of polyoxyethylated castor oil and 80% by volume of physiological saline, vortexed, and filtered to obtain a concentration of 1 mg. mL^-1Intravenous administration solution (as free base).

Preparation of a gastric lavage administration test solution of the product of Experimental example 11: about 180mg of Experimental example 11 was accurately weighed, respectively, and dissolved in 10% by volume of N, N-Dimethylacetamide (DMA), 10% by volume of polyoxyethylated castor oil and 80% by volume of physiological saline were added thereto, and vortexed to obtain a solution having a concentration of 1 mg. mL^-1Gavage dosing solution (as free base).

On the day of the experiment, after weighing the body weight, the theoretical administration volume of each beagle dog was calculated according to the following formula. The administration test solution should be prepared on the same day as the experiment. The actual dose and the time taken for the whole blood sample for each beagle dog were recorded in detail in the corresponding tables. The beagle dogs can recover to eat after being administrated for 4 hours, and can freely drink water in the experimental process.

Beagle dogs were fasted overnight the day before the experiment. On the day of experiment, beagle dogs in group A were injected with 1 mg/kg of the injection solution intravenously^-1(calculated as free base) of the solution of Experimental example 110.083, 0.25, 0.5, 1,2, 4, 8, 12 and 24 hours before and after administration, collecting blood by jugular vein, placing the blood in heparin sodium anticoagulation tube for anticoagulation, immediately and accurately sucking 200 microliter of whole blood, adding the whole blood into a test tube added with 600 microliter of acetonitrile for protein precipitation, carrying out vortex oscillation, and placing on wet ice. Stored in a refrigerator at-90 to-60 ℃ for biological sample analysis.

The beagle dogs in group B were gavaged with 5mg (based on free base) of the test solution of Experimental example 11, and 0.25, 0.5, 1,2, 4, 8, 12 and 24 hours before and after administration, and blood was collected from the jugular vein by 0.5mL, and after anticoagulation in a heparin sodium anticoagulation tube, 200. mu.L of whole blood was immediately and accurately aspirated, and the resulting sample was added to a test tube to which 600. mu.L of acetonitrile had been added for protein precipitation, vortexed, and placed on wet ice. Stored in a refrigerator at-90 to-60 ℃ for biological sample analysis.

The pharmacokinetic results are shown in figures 1 and 2 and the following table:

Claims (9)

1. a deuterium-containing azacyclic dione compound characterized in that: a compound represented by the following structural formula or a pharmaceutically acceptable salt thereof;

r1 is selected from: hydrogen, -C (═ O) R^A,-C(＝O)OR^A,-CH₂OC(＝O)R^A,-CH₂OC(＝O)OR^A,-CH(Me)OC(＝O)R^A,-CH(Me)OC(＝O)OR^A；

R^ASelected from alkyl, substituted alkyl;

R²and R³Identical or different, selected from hydrogen, alkyl, substituted alkyl;

R⁴selected from hydrogen, halogen, cyano, alkyl, substituted alkyl;

X¹,X²,X³,X⁴,X⁵,X⁶,X⁷,X⁸identical or different, selected from hydrogen, deuterium, halogen, cyano, nitro, amino;

Y¹,Y²,Y³identical or different, selected from hydrogen, deuterium;

said X¹,X²,X³,X⁴,X⁵,X⁶,X⁷,X⁸，Y¹,Y²,Y³At least one of which is deuterium.

2. A deuterium-containing azacyclic dione compound characterized in that: a compound represented by the following structural formula or a pharmaceutically acceptable salt thereof;

wherein R1 is selected from: hydrogen, -C (═ O) R^A,-C(＝O)OR^A,-CH₂OC(＝O)R^A,-CH₂OC(＝O)OR^A,-CH(Me)OC(＝O)R^A,-CH(Me)OC(＝O)OR^A；

R^ASelected from alkyl, substituted alkyl;

R⁴selected from hydrogen, halogen, cyano, alkyl, substituted alkyl;

n is a natural number of 0 to 5;

R₂' refers to any hydrogen atom on a cycloalkyl or heterocycloalkyl group being substituted with hydrogen, halogen, alkyl, substituted alkyl;

X¹,X²,X³,X⁴,X⁵,X⁶,X⁷,X⁸identical or different, selected from hydrogen, deuterium, halogen, cyano, nitro, amino;

Y¹,Y²,Y³identical or different, selected from hydrogen, deuterium;

said X¹,X²,X³,X⁴,X⁵,X⁶,X⁷,X⁸，Y¹,Y²,Y³At least one of which is deuterium.

3. A deuterium containing azaheterocyclic dione compound as claimed in claim 1 or 2, characterized in that:

the substituted alkyl refers to that one or more than one of hydrogen atoms on the alkyl is substituted by hydroxyl, halogen, cyano, nitro, amino, ether and ester.

4. A process for the preparation of a deuterium containing azaheterocyclic dione compound as claimed in claim 1 or 2, characterized in that:

the method comprises the following steps of (1): 1-5, carrying out substitution reaction on a substrate A and a substrate B under an acidic reaction condition to obtain a target deuterium-containing azacyclic diketone compound;

wherein the substrate A is a compound shown in the following structure:

substrate B is a compound represented by the following structure:

wherein when R is^1AIn the case of hydroxyl protecting groups, the target deuterium-containing azacyclic dione compound may also be deprotected to yield R^1AA product which is hydrogen;

when R is^1AIn the case of hydrogen, any reaction wherein hydrogen is substituted by a substituent may also occur at the hydroxyl group on the subject deuterium-containing azacyclic dione compound.

5. A process for the preparation of a deuterated azacyclic dione compound as claimed in claim 5, wherein: the target deuterium-containing azacyclic dione compound is subjected to a chiral separation procedure to obtain a chiral product.

6. A process for the preparation of a deuterated azacyclic dione compound as claimed in claim 5, wherein: the substrate A is prepared by the following steps: taking a derivative A0 of pyran formaldehyde as a raw material, and sequentially obtaining intermediate products A1, A2, A3, A4, A5, A6, A7, A8, A9 and A10 through reaction and later amine deprotection to obtain a substrate A;

wherein, the structures of the derivative A0 of the pyran formaldehyde and the intermediate products A1, A2, A3, A4, A5, A6, A7, A8, A9 and A10 are respectively as follows:

the derivatives of pyran carboxaldehyde A0 are

Intermediate A1 is

Intermediate A2 is

Intermediate A3 is

Intermediate A4 is

Intermediate A5 is

Intermediate A6 is

Intermediate A7 is

Intermediate A8 is

Intermediate A9 is

Intermediate A10 is

Wherein pro is a hydroxy protecting group;

pro' is an amine protecting group;

lea is a leaving group.

7. A deuterium containing azaheterocyclic dione compound as claimed in claim 1 or 2, characterized in that:

the substrate A is prepared by the following steps: taking a derivative A0 of pyran formaldehyde as a raw material, and reacting to sequentially obtain intermediate products A1-2, A2-2, A3-2, A4-2, A5-2, A6-2 and A7-2, and deprotecting rear amine groups to obtain a substrate A;

wherein, the structures of the derivative A0 of the pyran formaldehyde and the intermediate products A1-2, A2-2, A3-2, A4-2, A5-2, A6-2 and A7-2 are respectively as follows:

the derivatives of pyran carboxaldehyde A0 are

Intermediate A1-2 is

Intermediate A2-2 is

Intermediate A3-2 is

Intermediate A4-2 is

Intermediate A5-2 is

Intermediate A6-2 is

Intermediate A7-2 is

Wherein pro is a hydroxy protecting group;

pro' is an amine protecting group;

the corresponding carbon is R²、R³Substituted by a group, or by cycloalkyl, substituted cycloalkyl, heterocycloalkyl, or substituted heterocycloalkyl.

8. A deuterium containing azaheterocyclic dione compound as claimed in claim 1 or 2, characterized in that:

the substrate B is prepared by the following steps: halogenated benzene derivative B0 is used as a raw material, intermediate products B1, B2, B3 and B4 are obtained in sequence through reaction, and carbonyl is reduced into hydroxyl to obtain a substrate B;

wherein, the structures of the halogenated benzene derivative B0 and the intermediate products B1, B2, B3 and B4 are respectively shown as follows:

halogenobenzene derivative B0 is

Intermediate B1 is

Intermediate B2 is

Intermediate B3 is

Intermediate B4 is

Or

The intermediate product B4 can obtain an intermediate product B4-2 under the action of a deutero reagent, and then the intermediate product B is reduced into hydroxyl through carbonyl to obtain a substrate B;

the intermediate product B4-2 is a compound shown in the following structure:

9. a deuterium containing azaheterocyclic dione compound as claimed in any one of claims 1 to 9, characterized in that: is used as one of the components of a medicine or a medicine composition for treating influenza.

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